Abstract
Midbrain dopaminergic (mDA) neurons migrate to form the laterally-located substantia nigra pars compacta (SN) and medially-located ventral tegmental area (VTA), but little is known about the underlying cellular and molecular processes. Reelin signaling regulates tangential migration of SN-mDA neurons, but whether Reelin acts directly on SN-mDA neurons and how it affects their cellular morphology and migratory behavior has not been explored. Here we visualize the dynamic cell morphologies of tangentially migrating SN-mDA neurons with 3D-time-lapse imaging and identify two distinct migration modes. Slow migration is the default mode in SN-mDA neurons, while fast, laterally-directed migration occurs infrequently and is strongly associated with bipolar cell morphology. By speci1cally inactivating Reelin signaling in mDA neurons we demonstrate its direct role in SN-mDA tangential migration. We show that Reelin signaling promotes laterally-biased movements in mDA neurons during their slow migration mode, stabilizes leading process morphology and increases the probability of fast, laterally-directed migration.
Introduction
Dopaminergic neurons in the ventral midbrain (mDA neurons) are the major source of dopamine in the mammalian brain. Dysfunction in the dopaminergic system is associated with schizophrenia, addiction and depression, and degeneration of mDA neurons in the substantia nigra pars compacta (SN) results in the motor symptoms of Parkinson’s disease Grace and Bunney (1980), Volkow and Morales (2015), Przedborski (2017). mDA neurons originate in the 2oor plate of the ventral mesencephalon, from where they migrate to cluster into the laterally-positioned SN, the medially-located ventral tegmental area (VTA) and the posterior retrorubral 1eld. SN-mDA neurons project predominantly to the dorsal striatum and modulate voluntary movement Weisenhorn et al. (2016), while VTA-mDA neurons project to various forebrain targets, including the prefrontal cortex, nucleus accumbens and basolateral amygdala, and are important for the regulation of cognitive function and reward behavior Morales and Margolis (2017). How this anatomy is set up during development remains unclear.
mDA neurons differentiation starts at embryonic day (E) 10.5 in the mouse, when the 1rst mDA neurons that express tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis and a marker for differentiated mDA neurons, leave the ventricular zone of the ventral midbrain. Differentiated mDA neurons undergo a maturation process as they migrate to reach their 1nal positions Blaess and Ang (2015). We have previously shown that both SN- and VTA-mDA neurons undergo radial migration into the mantle layer of the developing ventral midbrain where they remain intermingled until E13.5. Between E13.5 and E15.5, mDA neurons destined for the SN migrate tangentially to more lateral positions, resulting in the segregation of mDA neurons into the laterally-located SN and the medially-situated VTA Bodea et al. (2014). This particular migration pattern suggests that SN-mDA neurons have the speci1c molecular machinery to respond to cues in their environment that direct their lateral migration. As exempli1ed by migration studies in cortical brain areas, a comprehensive characterization of migratory modes and accompanying changes in cell morphology is indispensable for unraveling the molecular mechanisms by which cell-type speci1c migratory behavior is regulated Kriegstein and Noctor (2004). So far, a detailed understanding of mDA neuronal migratory behavior has remained elusive due to challenges in visualizing migrating mDA neurons in suZcient detail.
At the molecular level, Reelin, an extracellular matrix molecule and known regulator of neuronal migration in various brain areas, is essential for the correct lateral localization of SN-mDA neurons. Reelin binds to its receptors APOER2 and VLDLR, and induces the phosphorylation of the intracellular transducer DAB1 Hiesberger et al. (1999), Trommsdorff et al. (1999). Phosphorylated DAB1 then mediates Reelin signaling by regulating cell adhesive properties or cytoskeletal stability Chai et al. (2016), Franco et al. (2011), Howell et al. (1997). In mice homozygous for null alleles of Reelin (reeler) or Dab1 (scrambler or Dab1null), in Vldlr/Apoer2 double knockout mice, or in organotypic slices in which Reelin signaling is blocked, SN-mDA neurons do not reach their 1nal positions in the ventrolateral midbrain and accumulate instead in the area of the lateral VTA Bodea et al. (2014), Vaswani and Blaess (2016) Kang et al. (2010); Nishikawa et al. (2003); Sharaf et al. (2013). Whether Reelin affects tangential (lateral) mDA neuronal migration directly, or whether the failure of SN-mDA neurons to reach their 1nal position in Reelin pathway mutants is due to alterations in glia 1bers or neighboring neuronal populations has not been explored. Moreover, it is not understood how the loss of Reelin signaling alters dynamic migration processes of mDA neurons and which of the multiple signaling events downstream of Reelin plays a role in mDA neuronal migration.
Here, we dissect the complex dynamic morphological changes that underlie the tangential migration of SN-mDA neurons using 2-photon excitation time-lapse imaging and a semi-automated data analysis pipeline. We 1nd that mDA neurons migrate in two modes: infrequent laterally-directed fast migration and frequent slow movement. We demonstrate that migrating mDA neurons undergo dynamic changes in cell morphology and show that fast, directed migratory spurts are strongly associated with bipolar morphology. Combining conditional gene inactivation, genetic fate mapping and time-lapse imaging, we demonstrate that Reelin affects mDA neuronal migration in a direct manner and promotes fast, laterally-directed migration of mDA neurons and stabilizes their leading process morphology.
Results
Reelin signaling acts directly on tangentially migrating mDA neurons
As a 1rst step to understand the regulation of mDA tangential migration by Reelin, we investigated whether Reelin signaling is directly required by mDA neurons for their correct lateral localization. We conditionally inactivated Dab1 in differentiated mDA neurons using a Cre-line in which Cre is knocked into the endogenous dopamine transporter (Dat) locus (genotype: DatCre/+, Dab1del/flox; referred to as Dab1 CKO) Ekstrand et al. (2007), Franco et al. (2011) (Figure 1A). To determine the onset of Cre-mediated recombination in the DATCre/+ mouse line, we crossed DAT loxCre/+mice with an enhanced yellow 2uorescent protein (YFP)-expressing reporter mouse line (Rosa26 lox−stop−lox−EY F P) Srinivas et al. (2001). We observed widespread YFP-expression in TH-positive (TH+) cells in the lateral mDA neuron domain starting at E13.5 (Figure 1-Figure supplement 1). Immunostaining for DAB1
SN-mDA neurons fail to migrate to their correct lateral position in reeler, Dab1 null or Apoer2/Vldlr double knock-out mutants Bodea et al. (2014), Howell et al. (1997), Nishikawa et al. (2003). To examine whether this phenotype is recapitulated in Dab1 CKO mice, we compared the mediolateral distribution of TH+ mDA neurons in coronal midbrain sections of control, Dab1 CKO and Dab1−/− (genotype: Dab1del/del ) mice at postnatal day (P)21-P30 and embryonic time points (E15.5 and E18.5)(Figure 1; Figure 1-Figure supplement 3). We focused our analysis on intermediate rostrocaudal sections of the TH+ mDA domain where the most severe defects in mediolateral distribution of mDA neurons are observed in reeler and Dab1−/− mice Bodea et al. (2014) (Figure 1B). In both the Dab1 CKO and Dab1−/− mice, mDA neurons failed to reach lateral positions in the SN and settled in more medial locations (Figure 1C-J; (Figure 1-Figure supplement 3). In addition, a few mDA neurons were aberrantly located dorsal to the VTA (Figure 1C-E, G-I). As the shift in the mediolateral distribution of mDA neurons observed in Dab1 CKO and Dab1−/− brains was similar, we conclude that Reelin acts directly on SN-mDA neurons to regulate their lateral migration.
We then asked whether such a direct function of Reelin is consistent with the localization of Reelin protein. During the time window of SN-mDA tangential migration (before E15.5), Reelin mRNA is expressed in the red nucleus, which is located dorsomedial to SN-mDA neurons. Whether Reelin protein is localized close to migrating SN-mDA neurons during this period has not been investigated Bodea et al. (2014), Nishikawa et al. (2003), Sharaf et al. (2015). Immunostaining for Reelin at E13.5 and E14.5 con1rmed strong expression of the protein in the region of the red nucleus (Figure 2B,C,E,F). At E13.5 and E14.5, Reelin protein, but not Reelin mRNA, was also observed ventral and lateral to the red nucleus, including the area where the most lateral mDA neurons are localized at these stages (Figure 2A-G). Thus, the localization of Reelin protein at E13.5-E14.5 is consistent with a direct role of Reelin signaling in SN-mDA neuronal migration.
Reelin signaling contributes to the segregation of SN- and VTA-mDA neurons into separate clusters
Given that SN-mDA neurons fail to form the lateral SN in the absence of Reelin signaling, we asked whether Reelin signaling is important for the segregation of SN- and VTA-mDA neurons into separate clusters. We have previously shown that mDA neurons positive for the potassium channel GIRK2 (G-protein-regulated inward-recti1er potassium channel 2; expressed in mDA neurons in the SN and lateral VTA) are shifted medially in Dab1−/− mice, while mDA neurons positive for Calbindin (expressed in VTA-mDA neurons and in a dorsal subset of SN-mDA neurons) are correctly localized Bodea et al. (2014), Björklund and Dunnett (2007). Comparison of the mediolateral position of TH+, Calbindin+ and TH+, GIRK2+ cells in control and Dab1 CKO brains at P30 showed that there was no signi1cant difference in the distribution of TH+, Calbindin+ mDA neurons between Dab1 CKO mice and controls (data not shown). In contrast, the TH+, GIRK2+ mDA subpopulation showed a signi1cant shift to a more medial position in the Dab1 CKO mice (Figure 3A-C). These results further con1rmed that the Dab1 CKO phenotype recapitulates the phenotype observed in Dab1−/− mice.
To investigate the distribution of medially shifted SN-mDA neurons within the VTA we analyzed the expression of the transcription factor SOX6 (sex determining region Y-box6), and the Lim domain protein LMO3 (LIM domain only protein 3) as markers for SN-mDA neurons and the expression of the transcription factor OTX2 (Orthodenticle homeobox 2) in VTA-mDA neurons Salvio et al. (2010), Panman et al. (2014), Poulin et al. (2014), Bifsha et al. (2017), Manno et al. (2016). In E18.5 control brains, TH+, OTX2+cells and TH+, SOX6+ cells were clearly separated at the boundary between SN and lateral VTA (Figure 3D,F). In Dab1 CKO mice, TH+, SOX6+ and TH+, Lmo3+ mDA neurons were more medially located than in controls and were partially intermingled with TH+, OTX2+ mDA neurons (Figure 3D-I). Hence, the inactivation of Reelin signaling in mDA neurons results in an ectopic medial location of SN-mDA neurons and a partial mixing of the two populations at what would constitute the SN-lateral VTA border in control brains.
Time-lapse imaging of tangentially migrating mDA neurons reveals diverse migratory behaviors across a population of neurons, and in individual neurons across time
Having established the direct requirement of Reelin signaling in the tangential migration of SN-mDA neurons, we visualized their migration in the presence and absence of Reelin, thereby dissecting out the precise migratory behaviors regulated by Reelin signaling. To monitor mDA migration during development, sparse labeling of SN-mDA neurons is necessary to enable tracking and morphology analysis of their migration. We used an established genetic inducible fate mapping system to mosaically label SN-mDA progenitors and their descendants Blaess et al. (2011), Bodea et al. (2014) (Figure 4A). With this system, SN-mDA neurons are preferentially labeled and more than two-thirds of YFP-labeled neurons are TH+ in the imaged regions at E13.5, and almost 90% are TH+ at E14.5 Bodea et al. (2014). Henceforth, we refer to these YFP-labeled neurons as SN-mDA neurons.
Ex vivo horizontal organotypic slice cultures of the ventral brain from E13.5 embryos with mosaically labelled SN-mDA neurons were prepared for time-lapse imaging Bodea and Blaess (2012); Bodea et al. (2014) (Figure 4B). 2-photon excitation time-lapse microscopy allows 3D visualization of dynamic changes in cell morphologies of migrating SN-mDA neurons. As the migratory modes and associated changes in morphology of tangentially-migrating mDA neurons are unknown, we 1rst de1ned migratory behavior in SN-mDA neurons using a number of parameters in slices of control mice and subsequently compared them with those of SN-mDA neurons in Dab1−/− slices.
To characterize the whole range of migratory behaviors within the time window of imaging, we acquired 3D volume images of slices every 10 minutes, and tracked soma positions of a large number of neurons (806 neurons from 3 control slices, 844 neurons from 3 Dab1−/− slices). We then calculated speed and trajectory for each neuron’s soma, at every time-point of imaging, based on location differences in consecutive volume images (Figure 4C-F, Movie 1). Plotting average speed distributions of cells from each slice, showed that the behavior of cells in different control slices and in different Dab1−/− slices was comparable (Figure 4-Figure supplement 1A,B). However, individual cells’ soma speeds varied considerably over time, and the maximum observed soma speed (henceforth max-speed) of a cell could be several times higher than its average speed (Figure 4F). Furthermore, ranking all control and all Dab1−/− cells by their max-speeds revealed great diversity as the max-speeds varied across cells in a smooth distribution from 183 µm/hr to 0 µm/hr for controls and from 134 µm/hr to 0 µm/hr for Dab1−/− cells (Figure 4-Figure supplement 1C,D).
Two modes of tangential migration in SN-mDA neurons: frequent, slow movements and infrequent, fast movements that are promoted by Reelin signaling
The role of Reelin signaling has been studied extensively in the cortex and hippocampus. However, only few studies have examined Reelin function in regulating the speed of migrating neurons. These studies have shown that the effect of Reelin varies depending on the brain region and type of neuron analyzed Simó et al. (2010); Britto et al. (2013), Britto et al. (2011), Wang et al. (2018). We have previously demonstrated that inhibiting Reelin in ex vivo slices results in a decrease in average speed of SN-mDA neurons over long periods of imaging Bodea et al. (2014). In our current analysis we found no signi1cant difference in the distribution of average speeds of the SN-mDA population in Dab1−/− slices compared to control slices (Figure 5A). However, distribution of max-speeds was signi1cantly shifted towards lower speeds in the absence of Reelin signaling (control: 25th percentile = 12.4 µm/hr, median = 23.6 µm/hr, 75thpercentile = 48.1 µm/hr, maximum = 183 µm/hr; Dab1−/−: 25th percentile = 10.1 µm/hr, median = 15 µm/hr, 75thpercentile = 29.8 µm/hr, maximum = 133.7 µm/hr) (Figure 5B).
We then asked whether this shift towards lower max-speeds in Dab1−/− SN-mDA neurons was accompanied by other changes in migratory behavior, or whether the neurons simply displayed lower max-speeds while maintaining the same migratory, directional and morphological characteristics as control SN-mDA neurons. To answer this question, we compared variation in soma speed over time, migratory direction and cell morphology of control and Dab1−/− mDA neurons with similar max-speeds. For this analysis, we divided control and Dab1−/− neurons into four groups based on the lower and upper quartiles of the Dab1−/− max-speed distribution. We de1ned these groups in the following manner: non-migratory cells with max-speeds of less than 10 µm/hr (control = 126/806, Dab1−/− = 205/844), ‘slow’ cells with max-speeds from 10-30 µm/hr (control = 355/806, Dab1−/− = 430/844), ‘moderate’ cells with max-speeds from 30-60 µm/hr (control = 186/806, Dab1−/−= 139/844) and ‘fast’ cells with max-speeds > 60 µm/hr, control = 139/806, Dab1−/− = 70/844) (Figure 5B). Non-migratory cells failed to move more than 1.7 µm in any two consecutive frames of analysis and were not included into the further analysis. Thus, a lower percentage of SN-mDA neurons reached moderate or fast migration speeds in Dab1−/− slices compared to controls, increasing the proportion of both non-migratory and ‘slow’ cells.
Next, we asked how frequently migrating SN-mDA neurons moved with soma speeds comparable to their max-speeds and whether the fraction of total time-points spent in high migratory speeds was different in control and Dab1−/− populations. To evaluate this, we used the criteria previously de1ned for max-speeds, but applied them to individual soma speeds for each cell at each time point. For example, we analyzed the fraction of time (percentage of total time-points) spent by each ‘fast’ cell with a soma speed of more than 60 µm/hr (fast migratory phase), 30-60 µm/hr (moderate migratory phase), 10-30 µm/hr (slow migratory phase) and less than 10 µm/hr (resting phase). In control slices, ‘fast’, ‘moderate’ and ‘slow’ cells spent a predominant fraction of time at rest (62.6 +/-20%; 68.5 +/-18.2%, 85.7 +/-11.1%, respectively) and were frequently in a slow migratory phase (26.8 +/-17.4%, 25.1 +/-16.3%, 14.2 +/- 11.1%, respectively). ‘Fast’ and ‘moderate’ cells achieved the moderate migratory phase in only a few frames (5.5 +/- 5.5% and 6.3 +/- 3.9, respectively), and the fast migratory phase (only in ‘fast’ cells) was equally infrequent (5.5 +/-2.2%) (Figure 5 B, Figure 5 - Figure supplement 1). The amount of time SN-mDA neurons of the same max-speed group spent in the resting phase or in the respective migratory phases was comparable between individual cells in control and Dab1−/− slices (Figure 5 - Figure supplement 1D-F).
In summary, these results demonstrate that SN-mDA migration has two distinct modes: a frequent slow migration phase seen in all migrating SN-mDA neurons and an infrequent moderate-to-fast phase occurring in a subset of SN-mDA neurons. These phases are superimposed over frequent periods of rest. Reelin signaling increases the proportion of migratory mDA neurons and the likelihood of moderate-to-fast movements in migrating mDA neurons. As moderate-to-fast migratory phases are only attained in very few frames in our slices, the average speed distribution of SN-mDA neurons are however not changed in Dab1−/− compared to control slices.
The Reelin-promoted infrequent fast movements of mDA neurons contribute to large directed cell displacements
We next asked whether max-speeds and directionality of migration were linked. We computed directionality as the ratio of total displacement (the 3D displacement between the initial and 1nal positions of the neurons) to path length (the distance travelled by each neuron summed up irrespective of direction) Petrie et al. (2009) for migrating SN-mDA populations in control and Dab1−/− slices. A high value of directionality (maximum value = 1) indicates almost no change in migratory direction while low values indicate frequent changes in direction. We found that directionality as well as total displacement generally increased with increasing max-speeds in SN-mDA populations from both control and Dab1−/− slices (Figure 5C-F; Figure 5 - Figure supplement 2). These data indicate that the infrequent moderate-to-fast movements in SN-mDA neurons result in major contributions to the directed migration of these cells. Since Reelin signaling increases the fraction of SN-mDA neurons that are able to undergo moderate-to-fast movements, Reelin supports directed migration of mDA neurons on a population level.
Reelin promotes preference for laterally-directed migration in mDA neurons
As tangential migration ultimately results in SN-mDA migration away from the midline, we analyzed the trajectories of migratory SN-mDA neurons in the presence and absence of Reelin signaling. We determined the “trajectory angle” for each cell as the angle between the midline (y-axis in live-images) and the cell’s displacement vector (Figure 6A). Thus, a trajectory angle of 900 indicates a cell whose total movement is precisely aligned to the lateral axis (x-axis in live-images). We de1ned a cell as migrating laterally if its trajectory angle was between 45 – 135°. We then evaluated the angular mean and standard deviation (σang;) for SN-mDA populations in control and Dab1−/− slices Berens (2009). We found that SN-mDA neurons from control slices displayed an anisotropy towards lateral migratory directions (mean 92.5°, σang; 68.4) while Dab1−/− SN-mDA neurons showed a signi1cantly reduced preference for lateral migration (mean 27.5°, σang 70.4) (see materials and methods for analysis of circular variables)(Figure 6B-D).
Next, to evaluate if ‘fast’, ‘moderate’ and ‘slow’ cell populations of control and Dab1−/− slices showed differences in their preference for lateral migration, we analyzed their trajectories separately. We found that trajectories of all three SN-mDA groups were anisotropic in controls, favoring migration towards lateral directions, but this anisotropy was greater in ‘fast’ and ‘moderate’ cells than in ‘slow’ cells (Figure 7A,D,G). Resolving this further into individual slow, moderate and fast migratory phases in the migratory mDA population, we also found that individual moderate-to-fast phases were more anisotropic than slow phases (Figure 7 - Figure supplement 1A,C,E).
In the absence of Reelin signaling, the trajectory pro1les of ‘slow’ neurons were signi1cantly altered with a complete loss of anisotropy towards lateral directions (mean −12.3°, σang 69.7°) (Figure 7B,C). In contrast, ‘moderate’ and ‘fast’ neurons still navigated to more lateral regions in Dab1−/− slices and their trajectory angle distributions were nearly identical to control neurons (Dab1−/−‘moderate’ neurons: mean 69.4°,σang58.7°; ‘fast’ neurons: mean 81°, σang 57.9°;) (Figure 7D-I). This 1nding also applies to slow, moderate and fast phases: slow phases are weakly laterally directed in controls, but in the absence of Reelin signaling individual slow migratory movements lose their slight lateral preference (Figure 7 - Figure supplement 1B,D,F and data not shown). These results show that Reelin signaling promotes lateral migration of SN-mDA neurons by increasing the fraction of SN-mDA neurons undergoing moderate-to-fast movements that are strongly biased for tangential movements and by promoting lateral anisotropy of ‘slow’ neurons.
mDA neurons adopt a bipolar morphology during moderate-to-fast phases of migration
Having thus de1ned the complex regulation of SN-mDA speed and trajectory pro1les by Reelin signaling, we investigated the cellular morphology that underlies mDA tangential migration. Since the dynamic cell morphologies of migrating SN-mDA neurons have not been assessed previously, we 1rst evaluated morphological changes in control SN-mDA neurons. Some cells had a stable, unbranched leading process (LP), and did not change their morphology, while other cells displayed dynamic LPs, that extended, retracted and branched frequently over time (Figure 8A-D; Figure 8 – Figure supplement 1; Movie 2).
We studied the cell morphology of SN-mDA neurons (70 ‘fast’, 40 ‘moderate’ and 40 ‘slow’ cells) in control and in Dab1−/− slices (49 ‘fast’, 40 ‘moderate’ and 40 ‘slow’ cells) and examined whether slow, moderate and fast migratory phases were associated with speci1c morphologies (for details of morphological analysis see materials and methods). We de1ned three morphological categories: a neuron was considered to be ‘bipolar-unbranched’ when a maximum of two processes arose directly from the soma and the LP was unbranched. Bipolar cells that extended a branched LP were de1ned as ‘bipolar-branched’. Neurons with more than two processes arising from the soma were de1ned as ‘multipolar’ (Figure 8A,C; Figure 8 – Figure supplement 1). The morphology of SN-mDA neurons evaluated based on YFP expression was indistinguishable from their morphology as assessed by TH-immunostaining in cleared whole-mount brains at E13.5 (Movie 3).
To investigate whether speci1c morphologies observed in SN-mDA neurons were associated with speci1c migratory speeds, we broke down the morphology of these cells into time points during which they were in bipolar-unbranched, bipolar-branched or multipolar phases (Figure 8 A,C, Figure 8 – Figure supplement 1) and paired their morphology with soma speed (as calculated by change in soma position between the current and the subsequent time point). Bipolarity was predominant in all phases of migration, but in both control and Dab1−/− SN-mDA neurons, fast and moderate migratory phases were almost exclusively associated with bipolar morphology. In contrast, about a third of slow migratory phases were associated with multipolar morphology. Hence, while slow migratory phases can occur in either bipolar or multipolar morphology, fast and moderate migration events are predominantly associated with bipolar morphology.
mDA neurons display unstable branch and leading process morphology in the absence of Reelin signaling
In time-lapse data-sets, some mDA neurons transitioned between bipolar and multipolar morphology, while others maintained either a bipolar or multipolar morphology during imaging. We next examined the proportions of migrating SN-mDA neurons that displayed a constant bipolar (branched and unbranched), constant multipolar or transitionary morphology over time (Figure 8A,C;Figure 8 – Figure supplement 1). This analysis enabled us to ask whether morphological stability is altered in the absence of Reelin signaling. In controls, transitionary cells made up about 40% of the total population. The proportion of transitionary cells was signi1cantly increased in the Dab1−/− population, while the population of bipolar neurons was decreased (Figure 9A, Table 1). Within the transitionary population, we found however no difference in the frequency of transitions between bipolar and multipolar morphologies for each neuron (de1ned as number of morphology
Finally, we randomly selected 20 control and 20 Dab1−/− mDA neurons with maximum soma speed of more than 10µm/hr and manually traced their morphology in 3D for the 1rst 19 imaging time-points (Figure 9 - Figure supplement 2). In all control and Dab1−/− mDA neurons, the LP remained stable and visible during the duration of imaging. We then compared the length of the LP (plus cell body) in control and Dab1−/− mDA neurons and found that mDA neurons in Dab1−/− slices displayed a broader distribution of LP length with very long and very short LPs (Figure 9 - Figure supplement 2G). Hence, in the absence of Reelin signaling, SN-mDA neurons display aberrant changes in morphology characterized by an increased proportion of transitionary neurons, an increase in unstable processes on the cell soma and LP and a greater variation in LP length.
Reelin downstream signaling in the ventral midbrain
As it is not known which downstream components of the Reelin signaling pathway regulate SN-mDA tangential migration, we investigated Reelin signaling events that were previously shown to in2uence neuronal polarity in migrating neurons in the cortex, hippocampus or spinal cord. Reelin signaling results in the activation (phosphorylation) of PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) through DAB1 Jossin and GoZnet (2007). PI3K activation results in phosphorylation (activation) of LIMK1 (Lim domain kinase 1) via Rac1/Cdc42 and PAK1. P-LIMK1 inactivates (phosphorylates) Co1lin1, an actin depolymerizing protein of the ADF/Co1lin family. Reelin-mediated inactivation of Co1lin 1 ultimately leads to the stabilization of the actin cytoskeleton and has been implicated in stabilizing LPs of radially migrating cortical neurons as well as in preventing the aberrant tangential migration of neurons of the autonomous nervous system in the spinal cord Maciver and Hussey (2002), Krüger et al. (2010), Franco et al. (2011), Chai et al. (2009); Frotscher et al. (2017). To detect a potential misregulation of these downstream events in absence of Reelin signaling, we performed immunoblotting on E14.5 embryonic ventral midbrain tissue for p-LIMK1/LIMK1 and p-Co1lin1/Co1lin1. We did not detect signi1cant differences in protein levels or in relative phosphorylation levels (Figure 10 and data not shown). Hence, we conclude that the regulation of LIMK1/Co1lin1 activity is unlikely to be the key event in controlling cytoskeletal stability in migrating mDA neurons downstream of Reelin signaling.
Next, we examined Cadherin2 (CDH2) expression in the ventral midbrain. Reelin signaling controls somal translocation of radially migrating cortical neurons by modulating cell adhesion properties through regulation of CDH2 via the Crk/C3G/Rap1 pathway Franco et al. (2011). Relative protein levels of CDH2 were similar in tissue lysates from control and Dab1−/− E14.5 ventral midbrain (Figure 10 - Figure supplement 1). Whether CDH2 levels are altered at the membrane of mDA neurons in Dab1−/− mice could not be assessed, since the immunostaining for CDH2 on sections was not of suZcient quality to make a clear assessment of changes in membrane localization.
Discussion
The correct tangential migration of mDA neurons is crucial for the formation of the SN. Our study provides the 1rst comprehensive insight into speed, trajectory and morphology pro1les of tangentially migrating mDA neurons, and uncovers the alterations of tangential migratory behavior that result in aberrant SN formation in the absence of Reelin signaling (Figure 11).
Reelin signaling directly regulates tangential migration of SN-mDA neurons
A number of previous studies established the importance of Reelin in the formation of the SN Kang et al. (2010), Nishikawa et al. (2003), Sharaf et al. (2013), Bodea et al. (2014), but it remained to be elucidated whether Reelin is directly required for the tangential migration of SN-mDA neurons. Studies in cortex have shown that while Reelin is directly required for the stabilization of the LP and for the orientation of radially-migrating cortical projection neurons Franco et al. (2011), Reelin also indirectly affects migration through regulating radial glia cell process extension, morphology and maturation Hartfuss et al. (2003), Keilani and Sugaya (2008). Tangentially migrating cortical interneurons are only indirectly affected by Reelin signaling: the improper cortical layering caused by defective radial migration in absence of Reelin signaling ultimately results in incorrect positioning of interneurons Yabut et al. (2007). Reelin also plays a role in interneuron precursors that undergo tangential chain migration to the olfactory bulb. However, it does not modulate tangential migration directly but rather acts as a detachment signal that regulates the switch form tangential chain migration to radial migration Hack et al. (2002). Evidence for a direct function of Reelin signaling in tangential neuronal migration comes from sympathetic preganglionic neurons in the spinal cord. In these neurons, Reelin has been shown to stabilize LPs via the phosphorylation of Co1lin 1 during tangential migration thereby preventing aberrant migration Krüger et al. (2010), Phelps (2002).
To explore whether Reelin has a direct role in tangential migration of SN-mDA neurons, we inactivated Dab1 in SN-mDA neurons starting at the onset of their tangential migration without affecting their earlier radial migration step and without inactivating Dab1 in other cell populations in the ventral midbrain. The similarity in mediolateral distribution of SN-mDA neurons in Dab1−/− and in Dab1 CKO implies that Reelin signaling has a direct effect on migrating SN-mDA neurons. We also con1rmed that the GIRK2-expressing mDA population, which consists of lateral VTA- and SN-mDA neurons was distributed in a similar manner than what we reported previously for Dab1−/− mice Bodea et al. (2014). Investigation of additional markers that label SN-mDA neurons more speci1cally, such as Lmo3 and SOX6, showed that the medially misplaced SN-mDA neurons were partially intermingled with VTA-mDA neurons. These results imply that in absence of Reelin signaling in mDA neurons, the separation of SN- and VTA-mDA neurons is not fully completed and SN-mDA neurons lose their ability to undergo the long-range tangential migration necessary to form the laterally-positioned SN. Thus, our 1ndings are the 1rst demonstration of Reelin as a direct regulator of tangential neuronal migration in the brain.
Reelin protein is localized in the lateral ventral midbrain
In the ventral midbrain, Reelin mRNA is restricted to the cells of the red nucleus at E13.5 and E14.5 Bodea et al. (2014) (Figure 2). Using immunostaining, we show that Reelin protein is distributed much more broadly at these stages. Strong labeling is seen in regions lateral to the migrating SN-mDA, while weaker staining is observed in the area where SN-mDA neurons are localized. Thus, the Reelin protein distribution that we describe here is consistent with a direct role of Reelin in regulating SN-mDA migration. Whether the red nucleus is the only source for Reelin in the ventral midbrain or whether there are additional sources remains to be investigated. Mouse mutants in which the RN is only partially formed do not show any obvious displacement of SN-mDA neurons (at least not up to E18.5), suggesting that other Reelin sources could be important for mDA migration Prakash et al. (2009). Reelin mRNA is expressed anterior to the SN, in the hypothalamus and ventral thalamus Alcántara et al. (1998). Moreover, it has been proposed that Reelin is transported from the striatum to the SN via axons in the striatonigral pathway Nishikawa et al. (2003). Indeed, Reelin is expressed in the early differentiating cells in the striatum, but the striatonigral pathway is probably only established (E17 in rat) after the critical time period for SN-mDA migration Fishell and Kooy (1987), Alcántara et al. (1998).
Reelin promotes the proportion of mDA neurons undergoing fast, directed migration
The visualization and tracking of a large population of migrating mDA neurons, and the subsequent categorization of the instantaneous soma speed of individual mDA neurons into slow, moderate and fast phases revealed that irrespective of their max-speed, mDA neurons spent a majority of their time at rest. During their migratory phase, mDA neurons move mostly at slow speed. Moderate-to-fast laterally-directed migration spurts that result in large displacements are infrequent and occur in only a subset of labeled mDA neurons during the time-window of imaging. Thus, mDA neurons migrate in two modes: in a frequent, slow mode and in infrequent, fast movements with a strong lateral orientation. A similar pattern of migration with variable instantaneous speeds and periods of rest has also been reported for newly generated granule cells in the dentate gyrus and for cortical projection neurons Simó et al. (2010), Wang et al. (2018).
Comparing mDA tangential migration in the presence and absence of Reelin signaling, we observed that the duration of the individual migratory phases as well as average speed distribution of mDA neurons was comparable between control and Dab1−/− slices, while the likelihood of moderate-to-fast migration events was decreased in mDA neurons in Dab1−/− slices. In addition, a higher proportion of mDA neurons spent the entire imaging period at rest. Hence, Reelin promotes the likelihood with which moderate-to-fast migration spurts occur and increases the proportion of cells that enter a migratory phase.
Interestingly, the increased presence of activated DAB1 in cortical projection neurons as a consequence of reduced ubiquitylation and degradation in absence of the E3 ubiquitin Ligase Cullin-5 leads to the opposite effect in the migratory behavior of these neurons: periods of rest are decreased and average as well as instantaneous speed is increased at late stages of cortical migration (E16.5) Simó et al. (2010). This would be consistent with the role of Reelin that we observe in the migration of mDA neurons. In contrast, average speed appears not to be altered in cortical neurons of reeler mutants at this stage of development Chai et al. (2016). Observation of cortical projection neurons in their multipolar-to-bipolar transition phase at E15.5 suggests yet another effect of Reelin: at this stage cortical neurons were observed to migrate faster in the absence of Reelin signaling while addition of exogenous Reelin slowed down migrating neurons, but only within the subventricular zone Britto et al. (2011), Britto et al. (2013). Thus, even in the same neuronal population, Reelin signaling might have diverse effects on the speed of neuronal migration at different stages of migration.
Reelin promotes a preference for directed migration
While moderate-to-fast migratory events are less likely in the Dab1−/− mDA population, individual moderate-to-fast Dab1−/− mDA neurons are equally laterally-directed as control mDA neurons. In contrast, slow cells, which are weakly anisotropic in controls are signi1cantly more isotropic in Dab1−/− slices. The loss of the laterally-directed slow movements might interfere with mDA neuron’s ability to initiate moderate-to-fast, laterally-directed spurts. Indeed, mDA neurons have an aberrant orientation in E13.5 reeler brains Bodea et al. (2014). In the cortex, Reelin regulates orientation and cell polarity of multipolar neurons in the intermediate zone facilitating their switch to bipolar, glia-dependent migration Gärtner et al. (2012), Gil-Sanz et al. (2013), Jossin and Cooper (2011). Cortical projection neurons in their early phase of migration have been shown to deviate from radial migratory trajectories, in the absence of Reelin signaling as well as in the presence of exogenous Reelin Britto et al. (2011), Britto et al. (2013), Chai et al. (2016). Reelin also promotes directionality during the radial migration of dentate gyrus cells Wang et al. (2018). Interestingly, a recent study provides evidence that mDA neurons derived from induced pluripotent stem cells homozygous or heterozygous for a REELIN deletion show a disruption in their directed migratory behavior in neurosphere assays. Since the disruption occurs in absence of any organized tissue structure, Reelin signaling seems to modulate the ability of mDA neurons for directed migration independently of a speci1c pattern of Reelin protein deposition in the surrounding tissue Arioka et al. (2018). In conclusion, Reelin appears to be a crucial factor in enabling SN-mDA neurons to initiate directed migration rather than a factor that guides SN-mDA neurons in a particular direction.
Reelin signaling promotes stable morphologies in SN-mDA neurons
We show that moderate and fast movements of mDA neurons are strongly associated with bipolar morphologies both in control and Dab1−/− slices. Bipolarity is still predominant in slow phases, but about a third of the slow phases are associated with a multipolar morphology. In control slices, more than half of mDA neurons maintain a bipolar morphology throughout the imaging period, while about 40% transition between multipolar and bipolar morphologies. Only a small subset of cells (about 10%) stays multipolar at all time points. In absence of Reelin signaling, the percentage of transitionary cells is signi1cantly increased, and the proportion of stable bipolar cells is decreased. Interestingly, the increase in the proportion of transitionary cells in Dab1−/− slices is particularly pronounced in the cell population that does not reach moderate-to-fast migration speeds and that is signi1cantly more isotropic (data not shown) suggesting a correlation between loss of anisotropy in these cells and increased transitioning between bipolar and multipolar morphology. In transitionary cells of Dab1−/− slices, there is a signi1cant increase in branch transitions at the soma and LP, a sign of decreased branch stability. Moreover, the length of the LP is signi1cantly more variable in Dab1−/− than in control neurons. Thus, Reelin signaling appears to promote stability of morphologies once they have been adopted at speci1c phases of migration in mDA neurons.
In cortical neurons, Reelin appears to have multiple effects on cell morphology. In dissociated cortical neuronal cultures, Reelin signaling results in an increase in 1lopodia formation, likely via activation of Cdc42 Leemhuis et al. (2010). Moreover,in presence ofexogenous Reelin in organotypic slice cultures, projection neurons in the ventricular zone display a greater proportion of multipolar morphology, a phenotype concomitant with reduced migratory speeds (see above, Britto et al. (2013)). In contrast, LP morphology of migrating cortical neurons is comparable in presence and absence of Reelin signaling when these neurons 1rst contact the marginal zone of the cortex, but Reelin signaling is required to maintain this morphology and a stable LP during the 1nal somal translocation step of these neurons Chai et al. (2016), Franco et al. (2011). Finally, a recent study showing the phosphorylation of DAB1 via the Netrin receptor deleted in colorectal cancer (DCC) has reported an increase in multipolar neurons in the subventricular zone of Dcc knockout cortex Zhang et al. (2018). In summary, depending on location, concentration, and sub-cellular localization, Reelin and DAB1 can have differing effects on the morphology of migrating neurons.
An indirect regulation of morphology by Reelin signaling has been reported in tangentially-migrating cortical interneurons. In interneurons, branching of LPs aids in precise sensing of the extracellular environment during chemotaxis Martini et al. (2008). In the inverted reeler cortex, interneurons display a signi1cantly higher number of branch nodes and higher length of LPs than interneurons in control brains Yabut et al. (2007). This aberrant morphology is accompanied by their ectopic location in cortical layers. Since interneurons do not directly require Reelin signaling for their migration, it is likely that their aberrant morphology in the reeler cortex is an indirect effect of their altered position. As we observe similar effects on cell morphology in Dab1−/− mDA neurons, the aberrant mDA neuronal morphology may be a consequence of an increased necessity to scan the environment for guidance cues in ectopic medial positions rather than a direct downstream effect of Reelin.
Reelin downstream signaling in SN-mDA neurons
It has previously been demonstrated that the regulation of CDH2 via the Crk/CrkL-C3G-Rap1 pathway at the cell surface is important for the effect of Reelin on the polarity of cortical projection neurons during their migration Franco et al. (2011), Park and Curran (2008), Sekine et al. (2012), Voss et al. (2008). Co1lin1 has been shown to stabilize the LPs of migrating cortical neurons downstream of Reelin signaling-activated LIMK1 Chai et al. (2016), Chai et al. (2009). However, we demonstrate here that expression and/or phosphorylation levels of these Reelin downstream effectors are not obviously altered in mDA neurons in the absence of Reelin signaling. Other signaling events that in2uence cortical migration downstream or in parallel to Reelin signaling are mediated through integrin a5ß1 or the Netrin1-DCC pathway. The knockdown of integrin a5ß1 in cortical neurons affects apical process stability during terminal translocation suggesting that additional adhesion molecules may be recruited by Reelin signaling Sekine et al. (2012). In the cortex, both CDH2 and integrin a5 ß 1 act downstream of Reelin, with integrin a5ß1 anchoring the leading tip of terminally translocating neurons in the marginal zone and CDH2 regulating the subsequent cell movements Sekine et al. (2014). Interestingly, integrin a5ß1 has been shown to be important for stabilizing neurite extensions of mDA neurons in vitro. Whether it plays a general role in stabilizing neuronal processes in mDA neurons, including LPs, and in mDA migration has not been explored Izumi et al. (2017). Recently, cross talk between Netrin1-DCC and Reelin–Dab1 pathways has been reported in migration of cortical projection neurons Zhang et al. (2018). The Netrin1–DCC pathway is also important for proper localization of SN-mDA neurons during development Li et al. (2014), Xu et al. (2010). Though the effect on mDA distribution induced by Dcc inactivation differs from the effect caused by Dab1 inactivation, it is possible that effectors downstream of the Netrin1-DCC pathway, such as focal adhesion kinase my play a role in mediating Reelin signal in mDA neurons Zhang et al. (2018).
Conclusion
Here we provide a detailed characterization of the migratory modes and cellular morphologies underlying SN-mDA tangential migration to gain a detailed understanding of SN formation. We demonstrate that Reelin directly regulates lateral, tangential migration of mDA neurons by stabilizing the morphology of mDA neurons, by promoting lateral anisotropy in small, slow movements and by increasing the frequency of laterally-directed moderate-to-fast migration events that cover larger distances. We thus present new mechanistic insight into how Reelin signaling controls tangential migration and regulates the formation of the SN and open the door to further investigations of the molecular mechanisms of mDA migration.
Methods and Materials
Mouse lines
Dab1floxand Dab1del mice Franco et al. (2011) were obtained from Dr. Amparo Acker-Palmer, University of Frankfurt. Dab1 CKO mice (genotype: DATCre/+, Dab1flox/del) were generated by crossing Dab1flox/flox mice with DATCre/+, Dab1+/del mice Ekstrand et al. (2007). Dab1del/+ mice were used to generate complete knockouts of Dab1 (Dab1−/−). DATCre/+ mice were crossed with ROSAloxP −STOP −loxP −EY F P mice (R26EY F P) Srinivas et al. (2001) to analyse the timing and extent of recombination. Mosaic labelling of migrating mDA neurons was achieved by crossing ShhCreER mice Harfe et al. (2004) with R26EY F P mice. Day of vaginal plug was recorded as E0.5. Mice were housed in a controlled environment, with 12-hour light/night cycles and ad libidum availability of food and water. All experiments were performed in compliance with the guidelines for the welfare of animals issued by the Federal Government of Germany and the guidelines of the University of Bonn.
Tamoxifen
Tamoxifen (75 mg/kg body weight) was administered by gavage to pregnant dams at E8.5 to label SN-mDA neurons Bodea et al. (2014). TM (Sigma Aldrich) was prepared as a 20 mg/mL solution in corn oil (Sigma Aldrich), with addition of progesterone (Sigma Aldrich, 5 mg/mL) to reduce miscarriages.
Immunohistochemistry
Pregnant dams were sacri1ced by cervical dislocation. Embryos were dissected in ice cold PBS. Heads (E13.5 – E15.5) or brains (E16.5 – E18.5) were 1xed in 4% paraformaldehyde (PFA) for 2 – 3 hrs at room temperature (RT). Adult mice were anesthetized with iso2uorane, perfused transcardially with phosphate buffered saline (PBS), followed by 4% PFA. Tissue was cryopreserved in OCT Tissue Tek (Sakura), embryonic tissue was cryosectioned at 14 µm, adult brains were cryosectioned at 40 µm thickness. Immunostaining was essentially performed as previously described Blaess et al. (2011).
For immunostainings, sections were 1xed brie2y in 4%PFA (5 min at RT), followed by 1 hr incubation in 10% NDS in 0.1% Triton in PBS (0.1% PBT). Sections were incubated overnight at 4°C in primary antibody in 3% NDS in 0.1% PBT. Sections were washed 3X in 0.1%-PBT and incubated for 2 h in secondary antibody in 3% NDS in 0.1% PBT before mounting with Aqua Polymount (PolysciencesInc.).
For the detection of SOX6, antigen retrieval was carried out in 0.1M EDTA for 30 min at 65°C before blocking, and Cy3-Streptavidin ampli1cation was used with biotinylated donkey anti-rabbit antibody. To improve detection of DAB1 with rabbit anti-DAB1 antibody in E15.5 embryonic sections, a tyramide signal ampli1cation (TSA) was carried out with the TSA kit (Perkin Elmer) as follows: Sections were blocked in the TSA kit blocking solution for 1 h followed by incubation with rabbit anti-DAB1 antibody (1:5000, Howell et al. (1997)) in 0.1% TBST (Tris buffered saline with 0.1% Triton) overnight at 4°C. After a washing step in TBST, sections were incubated for 2 h at RT with biotinylated donkey anti-rabbit in TBST, followed by another washing step and incubation with HRP conjugated Streptavidin (1: 1000) in TBST for 1 h at RT. Sections were again washed with TBST and incubated for 10 min with TSA detection reagent. After additional washing steps in TBST and 0.1% PBT sections were co-stained for TH following the standard immunostaining protocol. A complete list of primary and secondary antibodies is presented in Table 2.
Immuno blotting
WT and Dab1−/−embryos were prepped at E14.5. Ventral midbrain was isolated and snap-frozen in liquid nitrogen. Tissue extraction was performed with RIPA buffer (Sigma, R0278) supplemented with 1x Halt protease & phosphatase inhibitor (Thermo1sher Scienti1c, 78442) on ice according to the manufacturer’s instructions. Protein concentrations were determined by BCA assay (Thermo1sher Scienti1c) using a BSA calibration curve. Protein supernatant was mixed with 4x LDS buffer and loaded on a 4-12% Bis Tris gel (NuPAGE, NP0335BOX). Protein was blotted on a PVDF membrane, blocked for 1 h at RT and incubated with primary antibody overnight. After washing with TBST, membrane was incubated with a corresponding horse radish peroxidase (HRP) coupled secondary antibody. Membrane was washed with TBST and visualization of immunoreactive proteins was conducted with a chemiluminescent HRP substrate solution (Super signal femto, Thermo1sher Scienti1c/ Western HRP substrate, Merck Millipore) using a chemiluminescent imager (Chemidoc, Bio-Rad). Bound proteins were removed using 1x Western blot stripping buffer (2% SDS, 60,02 mM Tris (pH 6.8), 100 mM ß-mercaptoethanol) and immunodetection was repeated. For quanti1cation, densitometric analysis was performed, normalization was carried out with total protein (Amido Black, Sigma Aldrich) using the software Image Lab (Bio-Rad).
In situ hybridization
Sections were post-1xed in 4% PFA for 10 min, rinsed in PBS and acetylated in 0.1 M TEA (triethanolamine)-HCl with 125 µL acetic anhydride for 5 min with stirring. Sections were washed in PBS and brie2y dehydrated in 70%, 95% and 100% ethanol (EtOH). 1 µg of RNA probe was added to 1 mL hybridization buffer and incubated for 2 min at 80°C. Sections were air-dried and transferred to a humidi1ed hybridization cassette. A 1:1 mixture of formamide and H2O was used as humidifying solution. 300 µL hybridization solution containing RNA probe was added to each slide, slides were covered with RNase-free coverslips and incubated at 55°C overnight. On the following day, coverslips were removed in prewarmed 5X SSC. To reduce unspeci1c hybridization, sections were incubated in a 1:1 solution of formamide and 2X SSC (high stringency wash solution) for 30 min at 65°C. Sections were then washed with RNAse buffer, containing 0.1% RNase A at 37°C for 10 min to remove non-hybridized RNA. Sections were washed twice with high stringency solution for 20 min at 65°C, once with 2X SSC and once with 0.1X SSC for 15 min at 37°C. Sections were placed in a humidi1ed chamber and incubated with 10 % normal goat serum in 0.1% PBS-Tween (blocking solution) for 1 hour at RT. Sections were incubated with anti-DIG-AP Fab fragments (diluted 1:5000 in 1% goat serum in 0.1% PBS-Tween) for 3 h at RT, or overnight at 4°C. Sections were washed several times 0.1% PBS-Tween, followed by two washes in NTMT buffer (containing 1 mg/mL levamisole to reduce background of endogenous alkaline phosphatase activity) for 10 min at RT. Sections were incubated in BM purple, a substrate for alkaline phosphatase (with 0.5 mg/mL levamisole) at RT until signal was observed. The chromogenic reaction was stopped by a 10 min incubation in TE buffer at RT. Sections were then washed in PBS, and immunostained for TH.
Image acquisition of 1xed cryosections
Embryonic and adult sections were imaged at an inverted Zeiss AxioObserver Z1 microscope equipped with an ApoTome. Fluorescence images were acquired with Zeiss AxioCam MRm 1388 x 1040 pixels (Carl Zeiss). At 10X (EC PlnN 10x/0.3, Carl Zeiss) and 20X (EC PlnN 20x/0.5, Carl Zeiss) magni1cations, tile images were acquired with conventional epi2uorescence. ApoTome function was used to acquire tile images and z-stacks at 40X (Pln Apo 40x/1.3 Oil, Carl Zeiss) and 63X (Pln Apo 63x/1.4 Oil, Carl Zeiss) magni1cations. In situ hybridized sections were imaged with transillumination (AxioCam MRc, 1300 x 1030 pixels, Carl Zeiss) at the AxioObserver Z1 setup. Images were stitched with Zen blue software (Zeiss, 2012). Sections stained with Alexa 649 secondary antibody, and 63X confocal images were imaged at a Leica SP8 confocal microscope and stitched with Leica PC suite (Leica, 2014)
Organotypic slice culture and time lapse imaging
Organotypic slice cultures were generated as previously described Bodea and Blaess (2012). Slices were placed on Millicell membrane inserts (Merck) and incubated for 6-12 h at 37°C, 5% CO2, before imaging. Slices were brie2y examined at a Zeiss Axioobserver microscope with conventional epi2uorescence. Healthy slices, with well-de1ned, strongly 2uorescent cells, were chosen for twophoton excitation imaging. Slices on their membrane inserts were transferred to µ-Dish imaging dishes (Ibidi) containing 750 µL of prewarmed, fresh culture medium (5 mL Hank’s balanced salt solution, 9 mL DMEM high glucose (Sigma Aldrich), 5 mL horse serum, 200 µL Penicillin/Streptomycin for 20 mL of culture medium). Slices were imaged at 32X magni1cation (C-Achroplan 32x/0.85, Carl Zeiss) with an inverted, two-photon Zeiss LSM 710 NLO microscope, equipped with temperature and CO2 control (Pecon). The microscope setup and the 32X water immersion objective were preheated for 8 hours before time-lapse experiments. Images were acquired using 920 nm for excitation with a laser power of 5 - 10% (Laser: Chameleon UltraII, Coherent). A total of 3 control (ShhCreER/+, Rosa26lox−stop−loxY F P /+, Dab+/+ or ShhCreER/+, Rosa26lox−stop−loxY F P /+, Dab1del/+) and Dab1−/− slices (ShhCreER/+, Rosa26lox−stop−loxY F P /+, Dab1del/del), across 4 litters, were imaged as described. Of the 6 slices analyzed, 3 control and 2 Dab1−/−were imaged for 4.3 hours while one Dab1−/− slice was imaged for 2.6 hours. All imaged slices were post-stained with TH to con1rm that the region imaged was within the dopaminergic domain Bodea et al. (2014). Organotypic slice cultures were 1xed in 4% PFA for 1 h at RT, then rinsed in PBS and 0.3 % PBT for 10 min. Slices were incubated in blocking solution (10% NDS in 0.3% PBT) at RT for 2 h, or overnight at 4°C. After blocking, slices were incubated with primary antibody solution (3% NDS in 0.3% PBT) for 24 – 48 h at 4°C. The following primary antibodies and dilutions were used: rabbit anti-TH (1:500), rat anti-GFP (1:1000). Slices were washed in 0.3% PBT and then incubated in secondary antibody solution (3% NDS in 0.3% PBT), at RT for 4 h, or overnight at 4°C. Secondary antibodies donkey anti-rabbit Cy3 (1:200) and donkey anti-rat Alexa 488 (1:500) were used. All steps were carried out in a 6-well plate.
Immunostaining and clearing of whole mount embryonic brains
Brains from E13.5 embryos were 1xed in 4% PFA for 4 h at room temperature, or overnight at 4°C. Brains were washed with PBS, 0.3% PBT, and incubated with blocking solution (10%NDS in 0.3% PBT) overnight at 4°C. The brains were incubated with primary antibodies: rabbit anti-TH (1:500) and rat anti-GFP (1:1000) at 4°C for 2 days. Next, the primary antibody solution was removed and the brains were washed three times with 0.3% PBT at RT for 15 min. The tissue was incubated with secondary antibodies: donkey anti-rat IgG-DyLight 647 (1:100) and donkey anti-rabbit Cy3 (1:200) at RT for 1 day. Subsequently, the tissue was washed three times with 0.3% PBT and three times with PBS for 20 min. All washing steps and antibody solutions preparation were performed using 0.3 % PBT. All steps were carried out in 24-well plates.
Tissue clearing was carried out as described previously Schwarz et al. (2015). The procedure was modi1ed for embryonic tissue as described here: After immunostaining, brains were incubated in increasing concentrations (30%, 50%, 70%) of tert-butanol (pH 9.5) for 4 h at RT followed by 96% and 100% tert-butanol (pH 9.5) for 4 h at 33°C. Brains were then incubated overnight in a triethylamine pH-adjusted 1:1 mixture of benzyl alcohol/benzyl benzoate (BABB, pH 9.5) at 33°C. Brains were stored in clearing solution at 4°C and imaged within 1 week of clearing. Whole mount brains were imaged in clearing solution with a 20X BABB dipping objective (Olympus) at a Leica SP8 upright microscope. Multi-channel image acquisition of the whole brain (4-6 tiles, 150 – 200 z-steps, step-size = 1.5 µm) took 30 – 70 h, and resulted in multichannel datasets of large sizes (20 – 80 GB). Voxel size of thus acquired images was 0.432 µm X 0.432 µm X 1.5 µm. Individual tiles at each z-step were stitched together using the Leica SP8 PC suite (Leica, 2014).
Analysis of cell distribution in 1xed slices
Mediolateral distribution of mDA neurons was analyzed for n>3 animals at each time point of analysis (E15.5, E18.5 and P21-30) by constructing normalized bins spanning the entire TH-positive domain. Since we observed that in both, Dab1 CKO and Dab1 −/− mice, a few TH-positive cells of the lateral most SN lateralis were consistently present (yellow arrowheads (Figure 1D,E,H,I), we de1ned the mediolateral bins by quadrisecting a line extending from the midline to the lateral most TH positive cells (Figure 1B). The fraction of the total number of TH positive cells in each mediolateral bin was evaluated for control, Dab1 CKO and Dab1−/− brains.
Speed and trajectories of migrating mDA neurons
To prevent any bias in selection of cells for tracking, and to track a large number of neurons in 3D in our two-photon time lapse datasets, we used the semi-automatic plugin TrackMate in Fiji Tinevez et al. (2017). Before soma detection, a 3X3 median 1lter was applied by the TrackMate plugin, to reduce salt and pepper background noise. Soma detection was carried out using the Laplacian of Gaussian (LoG) detector in TrackMate. The soma detected by the TrackMate plugin were automatically linked across time, in 3D, by using the linear assignment problem (LAP) tracker in TrackMate Tinevez et al. (2017); Jaqaman et al. (2008). After automatic tracking, the track scheme view in TrackMate was used to check the accuracy of each track by eye. Spurious tracks were deleted and missed detections were added using the manual tracking mode in TrackMate. Excel 1les from the TrackMate plugin were imported into MatLab Wu et al. (2015). 3D soma velocity was obtained at every time point (in units of µm/hr) of the analysis (starting t = 10 min) as the change in soma position vector between the previous frame and the current frame, divided by the time duration (0.167 hr) between frames (code modi1ed from Wu et al. (2015)). This data was used to generate probability histograms for average soma speed, maximum soma speed, time spent at rest (de1ned as soma speed < 10 µm/ hr), time spent in slow migration (soma speed between 10 and 30 µm/ hr), time spent in medium-fast migration (30-60 µm/hr) and time spent in fast migration (soma speed > 60 µm/ hr). Categories for rest, slow, medium-fast and fast speeds were de1ned for the purpose of easy visualization of data, and were based on 25% percentile (10 µm/hr) and 75% percentile speeds (30 µm/hr) of Dab1−/− population.
Cell trajectory angles were measured in 2D as the angle between midline (positive y-axis in the image) and the line joining the 1rst and 1nal soma positions. Cells that moved with maximum speeds of less than 10 µm/hr were excluded from the trajectory analysis as they were categorized as being at rest. Statistics on trajectory angles were performed with CircStat: a MatLab toolbox Berens (2009).
Only cells for which the soma were detected at all time points of imaging were included in the analysis. Using this approach, we tracked 806 cells in slices from control mice (ShhCreER/+, Rosa26lox−stop−loxY F P /+, Dab+/+ or ShhCreER/+, Rosa26lox−stop−loxY F P /+, Dab1−/+) and 844 cells from Dab1−/− mice (ShhCreER/+, Rosa26lox−stop−loxY F P /+, Dab1−/−), across 3 slices and acquired their speed and trajectory pro1les. Each cell (and track) had a unique ID assigned by the TrackMate plugin. These cell IDs were used to identify and locate individual cells in the slice for further analysis.
Morphology analysis of migrating mDA neurons
We restricted our morphological analysis to n= 150 control (70 fast, 40 medium-fast and 40 slow cells), and 129 Dab1−/−(49 fast, 40 medium-fast and 40 slow) cells. We observed that Dab1−/− cells continuously extended neurites in slices and this made it diZcult to unambiguously assign processes to individual cells as imaging progressed. Hence, we examined the morphology of each cell, in 3D, for the 1rst 18 frames of imaging. Cell soma was de1ned as the spot detected/assigned to the cells in the TrackMate plugin. Analysis was done manually, by rendering individual neurons in 4D (3D projection over all time frames) in ImageJ, and recording the number of primary processes (arising from the soma) and secondary processes at each time point. A cell was de1ned as bipolar when fewer than two processes were observed arising directly from the soma. The appearance/ disappearance of any branch was regarded as a branch transition. At each time point, the morphology of the cell, and the number of branch transitions, was manually annotated to the spot position data of the cell in excel sheets exported from TrackMate. In addition, 20 control and Dab1−/− cells were randomly chosen for tracing in 3D. These neurons were traced manually in simple neurite tracer (SNT) plugin of Fiji Longair et al. (2011). Tracings were carried out, at each time point individually, for the 1rst 18 frames of imaging. Fills of traced neurons were generated semi-automatically in the SNT plugin. Fill thickness was decided by eye but was maintained across all time points for a cell. Maximum intensity projections were also generated for the 3D segmentation 1lls. SNT traces were also used to measure length of the leading process in 3D.
Statistical analysis
Statistical signi1cance of mediolateral distributions of TH+ mDA neurons in control, Dab1 CKO and Dab1−/− adult and embryonic brains were assessed by two-way ANOVA with Tukey’s correction for multiple comparisons (n =6 animals/ genotype, at P30 and n = 4 animals/ genotype at E18.5). At E15.5, mediolateral distribution of TH+ mDA neurons and P30 TH+ GIRK2+ mediolateral distributions in control and Dab1 CKO brains were assessed for statistical signi1cance by Student’s t-test. All non-parametric distributions were analyzed with Mann-Whitney’s non-parametric rank test, Kruskal-Wallis test or Kalmogrov-Smirnov test (mentioned in 1gure legends) in Prism 7/ MatLab. Circular variables were analyzed with the CircStat toolbox for MatLab. Angle distribution in populations were compared using Kuiper’s test for circular variables Berens (2009).
Manuscript submitted to elife
Movie 1. Time lapse imaging with 2 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 reveal aberrant orientation and slower migration of Dab1−/− mDA neurons.
Movie 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.
Movie 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 me-lapse experiments (E14.5) shows similar morphology with YFP (green) and TH (magenta) immunostaining.
Acknowledgements
This work was supported by the Maria von Linden-Program and BONFOR (both University of Bonn, to S.B.), the German Research Foundation (BL 767/2-1, BL 767/3-1 to S.B.), a German Academic Exchange Service doctoral fellowship (to A.R.V) and a European Union grant (FP7-HEALTH-F4-2013-602278-Neurostemcellrepair to O.B.). We thank Brian Howell for providing the Dab1 antibody; Walter Witke for providing the Co1lin1 antibody, Nils-Görran Larsson for the DATCre mouse line; Ulrich Müller for providing the Dab1del and Dab1flox mouse lines; Donato Di Monte and Michael Helwig for assistance with two-photon imaging; Jonas Doerr, Martin Schwarz and Anke Leinhaas for initial support with clearing and imaging of whole-mount brains; Petra Mocellin for initial support with image analysis; Killian Berendes for technical support; Gabriela Bodea, David Greenberg and Marianna Tolve for critical reading of the manuscript. The authors declare no competing 1nancial or non-1nancial interests.