The LIS1 protein is evolutionarily conserved from yeast to man and was first identified as one of the nuclear distribution mutants (NudF – for nuclear distribution F) in the filamentous fungus Aspergillus nidulans (Morris 2000; Xiang et al. 1995) amongst other mutants displaying also defective nuclear distribution including NudA, NudC and NudE (Efimov and Morris 2000; Xiang et al. 1999). Orthologues of Lis1 have been identified and cloned in many organisms including Drosophila (Liu et al. 2000) and mice (Hirotsune et al. 1997) whereby the murine LIS1 protein differs in only one aminoacid from the human version (Hirotsune et al. 1998; Reiner et al. 1993). In higher eukaryotes, LIS1 interacts with cytoplasmic dynein (NudA orthologue in A. nidulans) (Faulkner et al. 2000; Sasaki et al. 2000; Smith et al. 2000), which is critically involved in subcellular transport and directed cell movement (Kardon and Vale 2009). Lis1 interacts also with the two Nde1 and Ndel1 – formerly known a NudE and NudEL – paralogues that are homologous to A. nidulans NudE (Efimov and Morris 2000; Feng et al. 2000; Kitagawa et al. 2000; Niethammer et al. 2000; Sasaki et al. 2000; Smith et al. 2000). Structural analysis have revealed that both LIS1 and NDEL1 form homodimers (NDEL1 also tetramers) and that dimeric forms of NDEL1 are the principal configurations that interact with LIS1 (Bradshaw et al. 2009; Derewenda et al. 2007; Soares et al. 2012; Tarricone et al. 2004; Zylkiewicz et al. 2011). The interaction of LIS1 with NDE1 and NDEL1 critically influences the activity of cytoplasmic dynein (McKenney et al. 2010; Mesngon et al. 2006; Shmueli et al. 2010; Vallee et al. 2012; Yamada et al. 2008). In migrating cortical neurons, cytoplasmic dynein in concert with LIS1 act along microtubules to create forces, that eventually promote the saltatory nuclear movement toward the centrosome (Tsai and Gleeson 2005; Vallee et al. 2009). In fact, a recent model proposes that the Syne-1/2-SUN1/2 nuclear membrane proteins critically function in nucleokinesis by bridging the nucleus to the cytoskeleton via LIS1 and cytoplasmic dynein whereby the minus-end-directed microtubule motor dynein-dynactin complex then promotes nucleokinesis toward the centrosome (Zhang et al. 2009). This nucleus-centrosome coupling is the driving force and underlying molecular basis for the “two-stroke” model of neuronal migration (see also Chaps. 2, 4 and 7).

The regulation of the LIS1/NDEL1-complex and its functional interaction with dynein is controlled at multiple levels and it has been demonstrated that NDEL1 contains specific phosphorylation sites that are targeted by CDK5 (Niethammer et al. 2000) and Aurora A (Mori et al. 2007, 2009). NDEL1 is also regulated by palmitoylation on a conserved cysteine residue and it has been shown that palmitoylation of NDEL1 is essential for the function of cytoplasmic dynein and its downstream activities, including the control of cortical neuron migration (Shmueli et al. 2010). Besides posttranslational modifications regulating the LIS1/NDEL1-complex and thus dynein activity, NDEL1 (much more than LIS1) also serves as a structural and signaling platform for a large amount of interacting proteins [reviewed in (Chansard et al. 2011; Moon and Wynshaw-Boris 2013)].

The biochemical and biophysical studies described above resulted in a relatively detailed molecular model of how the LIS1/NDEL1-complex regulates dynein-mediated nuclear/somal migration (Tsai and Gleeson 2005; Vallee et al. 2009). In contrast, very little is known how the LIS1/NDEL1-complex orchestrates neuronal migration during cortical development in vivo. Lis1 and Ndel1 are essential genes and complete ablation of Lis1 and Ndel1 in knockout mice results in early embryonic lethality at E5.5 pre-implantation stages (Cahana et al. 2001; Hirotsune et al. 1998; Sasaki et al. 2005), and thus complicating the functional analysis of Lis1/Ndel1 function in vivo. Besides controlling neuronal migration, Lis1 has also essential functions in cell proliferation and neurogenesis which further compromises loss-of-function analysis of LIS1 during cortical neuron migration (Faulkner et al. 2000; Hippenmeyer et al. 2010; Tsai et al. 2005; Yingling et al. 2008). However, the dose of Lis1 is clearly very important and reduced levels of LIS1 in mice results in defects in the radial migration of several different types of neurons including cortical projection neurons (Cahana et al. 2001; Gambello et al. 2003; Hippenmeyer et al. 2010; Hirotsune et al. 1998). Systematic examination of the consequences of dosage reduction of LIS1 on cortical development was achieved by comparing wild-type (Lis1 ^+/+ ), null heterozygotes (Lis1 ^ko/+ ), compound heterozygote (null/hypomorph, Lis1 ^ko/hc ), and conditional null (Lis1 ^ko/hc ;Cre) and it was found that Lis1 likely controls the efficiency of neuronal migration in a dose-dependent manner (Cahana et al. 2001; Gambello et al. 2003; Hippenmeyer et al. 2010; Hirotsune et al. 1998; Youn et al. 2009). It remains unclear from these genetic in vivo studies whether Lis1 has specific functions at discrete steps in the radial migration process. In contrast, in utero electroporation of Lis1 small interference RNA and short hairpin dominant negative Lis1 in wild-type background in rat and mouse brains caused a dramatic accumulation of multipolar cells in the SVZ (Shu et al. 2004; Tsai et al. 2005). In addition, the above Lis1 knockdown experiments indicate that even bipolar neurons in the IZ critically depend on Lis1 function for locomotion, presumably by regulating nuclear translocation through centrosome-nucleus coupling (Shu et al. 2004; Tsai et al. 2005; Tsai and Gleeson 2005; Vallee et al. 2009). These findings are also in line with the total inhibition of migration phenotype observed in cortical slice assays upon complete loss of Lis1 in conditional null (Lis1 ^ko/hc ;Cre) (Youn et al. 2009). We have recently applied the MADM (Mosaic Analysis with Double Markers) strategy (Zong et al. 2005) to ablate Lis1 in sparse subpopulations of cortical projection neurons (Hippenmeyer et al. 2010). In the experimental MADM paradigm, homozygous mutant cells are labeled in one color (e.g. green by GFP), wild-type cells in another color (e.g. red by tdTomato) and heterozygous cells in yellow (i.e. green and red together) in an otherwise unlabeled background (Hippenmeyer et al. 2010; Zong et al. 2005). MADM-based functional gene analysis has the advantage that essential genes can be studied at any stage during development since the sparseness of gene knockout in single cells allows the bypass of embryonic lethality. Consequently, we have quantified the distribution of Lis1 ^+/+ , Lis1 ^+/− and Lis1 ^−/− cortical projection neurons at adult stages and noticed a significant reduction of Lis1 ^+/− and Lis1 ^−/− neurons in the uppermost layers II/III but concomitant increase in lower layers. Interestingly, there was no significant difference between Lis1 ^+/− and Lis1 ^−/− in their laminar distribution although the migration of Lis1 ^−/− neurons is (at least initially) significantly delayed at birth when compared to Lis1 ^+/− . In contrast, Lis1 ^+/+ neurons displayed a significant cell-autonomous migration advantage when compared to Lis1 ^+/− and Lis1 ^−/− neurons, respectively. The presence of MADM-labeled Lis1 ^−/− neurons in all cortical layers indicates that non-autonomous and/or community effects could strongly influence the positioning of these Lis1 ^−/− mutant (and heterozygous Lis1 ^+/− ) neurons (see also below), given the critical function of Lis1 in promoting nuclear/somal translocation. Future detailed MADM-based analysis of Lis1 at embryonic stages may promise further insight into the cell-autonomous and possibly cell-nonautonomous functions of Lis1 in regulating the sequential steps of cortical neuron migration. In summary, Lis1 acts in a dose-sensitive manner to regulate the efficiency of neuronal migration, likely by promoting somal translocation via nucleus-centrosome coupling in migratory neurons and catalyzes the exit from the multipolar conformation to the bipolar state of nascent neurons in the embryonic SVZ (Fig. 1.3a). Experiments involving mosaic ablation of Lis1 by MADM in overall heterozygote Lis1 ^+/− mice also indicate a significant degree of intersecting community effects (upon reduction of Lis1) and/or cell-nonautonomous function(s) for Lis1 in regulating neuronal migration (Hippenmeyer et al. 2010; Youn et al. 2009).

Like for Lis1, homozygous complete knockout of Ndel1 in mice results in embryonic lethality (Sasaki et al. 2005) and although heterozygote Ndel1 ^+/− mice do not show obvious phenotypes, further reduction of Ndel1 in Ndel1 ^ko/hc (null/hypomorph) animals results in mild neuronal migration phenotypes (Sasaki et al. 2005; Youn et al. 2009). Ndel1 RNAi knockdown experiments by in utero electroporation independently revealed an essential function for Ndel1 for appropriate cortical projection neuron migration (Shu et al. 2004). The function of Ndel1 involves proper coupling of the centrosome to the nucleus, presumably in a Lis1 dependent manner since the requirement for Ndel1 in cortical neuron migration could be partially compensated by overexpression of Lis1 (Shu et al. 2004). Complete loss of Ndel1 in conditional knockout mice (Ndel1 ^ko/hc ;Cre) led to total inhibition of neuronal migration in organotypic cortical brain slices as revealed by high resolution time-lapse two-photon videomicroscopy (Youn et al. 2009). However, by using the same migration assay, reduction of NDEL1 protein levels to about 35 % in Ndel1 ^ko/hc allowed these neurons to migrate, albeit at reduced speeds. The slower migrating neurons in Ndel1 ^ko/hc displayed multiple branches, like multipolar cells, and exhibited a branched migration pattern (Youn et al. 2009). Such branched migration is very similar to the movements of neurons lacking p35 (Chae et al. 1997; Gupta et al. 2003), which is the activator of CDK5 (Tsai et al. 1994). In p35 ^−/− mutant mice (Chae et al. 1997) the vast majority of radially migrating cortical projection neurons displayed branched migration behavior (Gupta et al. 2003). Importantly, p35/CDK5 phosphorylates NDEL1 at specific sites (S198, T219 and S231) and this phosphorylation is essential for NDEL1 function (Niethammer et al. 2000; Sasaki et al. 2000). Therefore it is likely that the similar neuronal migration phenotypes of neurons lacking p35 or having reduced levels of NDEL1 could be causally related. Whether p35/CDK5-mediated NDEL1 phosphorylation is critically required during the entire radial neuron migration process or just at discrete steps is an important open question. In fact, loss of CDK5 function results in the accumulation migrating cortical projection neurons in the IZ and many of these Cdk5 ^−/− mutant neurons remain in a multipolar configuration (Gilmore et al. 1998; Ohshima et al. 1996, 2007). Thus, p35/CDK5-induced phosphorylation of NDEL1 could in principle, besides promoting migration, trigger the multi-to-bipolar transition although distinct pathways including other CDK5 target molecules likely act in parallel in these sequential radial migration steps.

In order to get further insights and identify the critical step(s) in cortical neuron migration controlled by NDEL1 function in vivo, we pursued functional MADM analysis of Ndel1 (Hippenmeyer et al. 2010). By using developmental time course and single clone analyses, the phenotype of mutant Ndel1 ^−/− MADM-labeled cells revealed that Ndel1 cell-autonomously regulates a very specific step in cortical neuron migration: the entry into the CP (Fig. 1.3b). Live-imaging experiments of MADM-lableled neurons in organotypic slice preparations further indicated that the speed of migration for Ndel1 ^+/+ and Ndel1 ^−/− neurons is not significantly different as long as the neurons migrate within the IZ. In contrast, the majority of Ndel1 ^−/− neurons failed to cross the IZ-CP border (Fig. 1.4). Altogether, the MADM experiments showed that Ndel1 ^−/− neurons in a mosaic, mostly heterozygous, environment can migrate through the VZ/SVZ and the entire IZ. However, in the MADM setting, Ndel1 ^−/− neurons are unable to enter and/or migrate into their CP target lamina. The analysis of several different types of MADM-labeled Ndel1 ^−/− mutant neurons, besides cortical projection neurons, indicates that NDEL1 is generally required for the invasion of the target area in the course of radial neuronal migration (Hippenmeyer et al. 2010). The mosaic analyses afforded by MADM not only revealed cell-autonomous functions of Ndel1 (and Lis1, see above) but indicates a significant amount of cell-nonautonomous effects which critically contribute to the regulation of discrete steps in radial neuron migration. Most indicative for such cell-nonautonomous effects is the fact that the phenotypes resulting from sparse MADM-based gene knockout are distinct from those observed in whole cortex knockout (Hippenmeyer et al. 2010; Youn et al. 2009) (Fig. 1.4). For instance, Ndel1 ^−/− mutant neurons were unable to move in cultured organotypic brain slices from Ndel1 ^ko/hc ;Cre mice (complete loss of NDEL1 in cortex) but MADM-labeled Ndel1 ^−/− neurons (in genetic mosaic, mostly normal environment) could migrate at regular speeds in IZ and only stalled once they reached their CP target area. Thus, it is conceivable that the inability of Ndel1 ^−/− mutant neurons to migrate in Ndel1 ^ko/hc ;Cre mice is potentially a consequence of adversive cell-nonautonomous effects. What could be the molecular nature of such ‘negative’ effects? Neuronal migration requires the dynamic adjustment of cell adhesion molecules such as N-cadherin (see also below) and possibly axon guidance molecules (see also Chap.​ 9). Thus, one could speculate that the overall molecular landscape at the cell membrane might be non-permissive for migration and/or even inhibiting when Ndel1 ^−/− neurons contact each other. Conversely, in a mosaic MADM environment, most Ndel1 ^−/− mutant neurons only get in contact with Ndel1 ^+/− or Ndel1 ^+/+ neurons and cross-inhibiting Ndel1 ^−/− – Ndel1 ^−/− cell contacts are minimized. On the other hand, migrating MADM-labeled Ndel1 ^−/− neurons, as long as progressing through the VZ/SVZ and the IZ, could even be positively influenced by the neighboring Ndel1 ^+/− or Ndel1 ^+/+ neurons. Such ‘positive’ effects might also account for the positioning of MADM-labeled Lis1 ^−/− in upper cortical layers (see above). The precise nature of the ‘positive’ cell-nonautonomous effects and how they account for positioning and/or migration of mutant Ndel1 ^−/− and Lis1 ^−/− cortical projection neurons is currently unclear. In principle two scenarios could be possible: (1) community effects whereby isolated Ndel1 ^−/− or Lis1 ^−/− neurons, despite defective in the intrinsic cytoskeletal migration machinery (e.g. nucleus-centrosome coupling), may still “piggyback” on normally migrating abutting neurons; and (2) neurons migrating in a crowd could actively signal, in a Lis1/Ndel1-dependent manner, to each other in order to boost the intrinsic migration machinery. Molecularly, such a mechanism poses however a significant challenge since intracellular cytoplasmic proteins would need to signal inside-out and trans-cellular to neighboring migrating cells. Such a signaling pathway likely would need to involve transmembrane receptors and possibly extracellular matrix components, and some sort of a sensor connecting to cytoskeletal force generators in the ‘signal-receiving’ cell. Interestingly, cell-on-cell migration of Drosophila invasive border cells, which also depends on cell-autonomous requirement for dLis1, has been proposed to involve “piggy-backing” (Yang et al. 2012). More generally, community effects have been observed in a variety of other cell types moving collectively (Friedl and Gilmour 2009; Tada and Heisenberg 2012; Theveneau and Mayor 2013).

The adaptor protein 14-3-3ε is encoded by YWHAE in human and deleted in all MDS patients (Cardoso et al. 2003; Toyo-oka et al. 2003; Wynshaw-Boris 2007). The precise function of 14-3-3ε during brain development is unclear but it has been shown that Ywhae genetically interacts with Lis1 in mice and that the 14-3-3ε protein binds to p35/CDK5 phosphorylated NDEL1 (Toyo-oka et al. 2003). The 14-3-3ε/NDEL1 interaction is important for binding to LIS1 and the dynein motor. Thus, the tripartite LIS1/NDEL1/14-3-3ε complex appears as a key regulator of neuronal migration. Interestingly, sparse MADM-mediated conditional knockout of Ywhae in cortical projection neurons did not significantly affect their radial migration although Ywhae ^−/− pyramidal CA1 cells in hippocampus showed slight defects in migration (Hippenmeyer et al. 2010). Since multiple isoforms of 14-3-3 are expressed in the brain (Takahashi 2003), distinct 14-3-3 isoforms could compensate for the loss of 14-3-3ε function. Along the same lines, genetic redundancy could also, at least in part, explain some of the divergent phenotypes that were observed when comparing the individual MADM-induced Lis1 ^−/− , Ndel1 ^−/− and 14-3-3ε ^−/− mutant neurons to each other. For instance Nde1, the second homologue of A. nidulans NudE and paralogue of murine Ndel1, shares 55 % sequence homology with Ndel1 (Feng et al. 2000; Sweeney et al. 2001) and could possibly compensate for some Ndel1 functions, especially during the early stages of neuronal migration in VZ/SVZ and IZ. Although the overall phenotype of Nde1 ^−/− mutant mice is distinct from Ndel1 ^−/− mutants, NDE1 can interact with LIS1 and this interaction is critical to control cortical lamination and thus cortical neuron migration (Feng et al. 2000; Feng and Walsh 2004; Pawlisz et al. 2008). Alternatively, LIS1, NDEL1 and 14-3-3ε may also each act in a ‘complex’-independent manner to execute distinct component-specific functions, at discrete steps and transitions during radial migration of cortical projection neurons. Recent biochemical experiments support such a model whereby for example both LIS1 and NDEL1 proteins may act independently in certain contexts and that their interaction can amplify and/or modulate those activities (Zylkiewicz et al. 2011).