Morphogenesis of the central nervous system relies in large part upon the correct migration of neuronal cells from birthplace to final position. Two general modes of migration govern CNS morphogenesis: radial, which is mostly glia-guided and topologically relatively simple; and tangential, which often involves complex movement of neurons in more than one direction. We describe the consequences of loss of function of presenilin 1 on these fundamental processes. Previous studies of the central nervous system in presenilin 1 homozygote mutant embryos identified a premature neuronal differentiation that is transient and localized, with cortical dysplasia at later stages. We document widespread effects on CNS morphogenesis that appear strongly linked to defective neuronal migration. Loss of presenilin 1 function perturbs both radial and tangential migration in cerebral cortex, and several tangential migratory pathways in the brainstem. The inability of cells to execute their migratory trajectories affects cortical lamination, formation of the facial branchiomotor nucleus, the spread of cerebellar granule cell precursors to form the external granule layer and development of the pontine nuclei. Finally, overall morphogenesis of the mid-hindbrain region is abnormal,resulting in incomplete midline fusion of the cerebellum and overgrowth of the caudal midbrain. These observations indicate that in the absence of presenilin 1 function, the ability of a cell to move can be severely impaired regardless of its mode of migration, and, at a grosser level, brain morphogenesis is perturbed. Our results demonstrate that presenilin 1 plays a much more important role in brain development than has been assumed, consistent with a pleiotropic involvement of this molecule in cellular signaling.

Presenilin 1 and 2 (Psen1 and Psen2) are polytopic membrane proteins that are mutated in the majority of pedigrees with early-onset familial Alzheimer’s disease (Price and Sisodia, 1998). Compelling evidence has accumulated supporting a role for Psen1 in intramembranous, ‘γ-secretase’ processing not only of the β-amyloid precursor protein (APP)(De Strooper et al., 1998; Naruse et al., 1998), but also an increasing list of various type I membrane proteins, including Notch1(De Strooper et al., 1999; Struhl and Greenwald, 1999),Erb-B4 (Ni et al., 2001), N-and E-cadherins (Marambaud et al.,2002), low density lipoprotein receptor-related protein(May et al., 2002), CD44(Lammich et al., 2002), nectin 1a (Kim et al., 2002), and DCC(Taniguchi et al., 2003) (for a review, see Sisodia and St George-Hyslop, 2002). Although it has become clear that the role of Psen1 reaches beyond the processing of APP, the exact molecular rules underlying its involvement in other processes, or the possible interdependence of these processes, are not completely understood. For example, although Psen1 is important for processing of Notch, which is itself indispensable for development beyond day 10 of embryogenesis(Swiatek et al., 1994; Conlon et al., 1995), Psen1 homozygote mutant embryos (Psen1 mutants) only die perinatally (Wong et al.,1997; Shen et al.,1997). Psen1 mutants have defects in axial skeletal development and in neuronal differentiation(Wong et al., 1997; Shen et al., 1997; Hartmann et al., 1999; Handler et al., 2000; Yuasa et al., 2002), but Psen2 apparently compensates for some of the embryonic functions of Psen1. Psen1/Psen2 double mutant embryos die before day 9.5 with severe patterning defects (Donoviel et al., 1999; Herreman et al.,1999).

In the present study, we asked if the emerging complexity of the biochemical function of presenilin is reflected in the phenotype of Psen1 mutants (Wong et al.,1997), focusing on CNS morphogenesis and neuronal migration.

One of the best-studied examples of radial migration is the formation of the cerebral cortex (Marín and Rubenstein, 2003; Rakic,2003). Molecular and cellular studies coupled with analyses of natural and targeted mutations in the mouse have indicated that radial migration is under the control of at least two signaling pathways, the one involving reelin and its receptors (Tissir and Goffinet, 2003), the other dependent upon Cdk5 and its regulatory subunits p35 and p39 (Dhavan and Tsai, 2001; Ohshima and Mikoshiba, 2002). Tangential migration, however, is a broad term used to group together various forms of neuronal movement along the anteroposterior or dorsoventral axis of the neural tube. Paradigms of tangential migration include interneurons migrating from the ganglionic eminences into the cortex and the olfactory bulb; neuronal precursors of the cerebellum and the precerebellar system migrating from the rhombic lip; and facial motoneurons migrating within the brainstem. Tangential migration has been associated with a host of molecular signals, including motogenic factors,extracellular matrix and cell-adhesion molecules, and many of the same chemoattractive and chemorepulsive signals implicated in axon guidance(Marín and Rubenstein,2001; Marín and Rubenstein, 2003).

We examined the development of the cerebral cortex as an example of both radial and tangential migration, and hippocampal dentate gyrus precursors, the external granular layer of the cerebellum, the precerebellar system and facial branchiomotor neurons as varied examples of tangential migration. In addition to widespread neuronal migration defects in the cortex, hippocampus, midbrain,cerebellar system and hindbrain, we documented defects in morphogenesis of the mid-hindbrain region. We found that both general modes of migration are disturbed in the Psen1 mutants, suggesting that the many disparate molecular mechanisms directly or indirectly governing neuronal migration are simultaneously affected.

Mice

The Psen1 mutation (Wong et al., 1997) was maintained on C57BL/6 background. A total of 34 litters between E10.5 and E17.5 (155 wild-type or heterozygous and 65 homozygous embryos) were analyzed.

Retrograde labeling and photoconversion

Embryos were fixed overnight in phosphate-buffered 4% paraformaldehyde. A solution of DiI (Molecular Probes) (0.5% in ethanol, further diluted 1:10 in 0.3 M sucrose) was injected into the VIIth cranial nerve root exposed from the ventral aspect after dissection; DiO (Molecular Probes) was injected into the medial mass in the mutants. To label pia-attached radial glia, DiI crystals were placed along the pia. Photoconversion of DiI into a stable product was performed as described (Louvi and Wassef,2000).

BrdU labeling

For labeling of dividing cells at E17.5, pregnant females were injected intraperitoneally with a solution of BrdU (15 mg/ml in saline) at 20 μg/g of body weight and sacrificed 4 hours later. BrdU incorporation was detected with anti-BrdU-FITC antibody (Beckton-Dickinson) as described(Tole et al., 1997).

In situ hybridization and immunohistochemistry

Both were performed as described (Louvi and Wassef, 2000). RNA probes used were for the following genes:β-tubulin (type III), Cdh6, Cdh8, Dab1, Dlx2, ephrin A5,F-spondin, Gad67, Gata3, Gbx2, Hes5, Hoxb1, Islet1, Lmx1a, Lmx1b, Math1,NeuroD, Pax6, Phox2b, p75, Prox1, reelin, Rora, Scip, Tag1, Tbr1and Th. RC2 mAb (1:2) was from Developmental Studies Hybridoma Bank.

Defects in facial branchiomotor neuron migration

Facial branchiomotor (FBM) neurons are born in ventral rhombomere (r) 4 and undergo complex migration before they reach their final destination close to the pial surface of r6, where they form the facial motor nucleus(Altman and Bayer, 1982). Observation of Psen1 mutants revealed a cellular mass, accumulating medially on the ventricular surface of the ventral hindbrain from embryonic day (E) 11.5 onwards. This mass consisted of postmitotic neurons, as indicated by class III β-tubulin in situ hybridization and anti-neurofilament immunofluorescence; TUNEL assay revealed no differences in cell death in this area in comparison with wild-type embryos(Fig. 1A,B; data not shown). Floor-plate markers, including Shh and netrin, were unaffected and inter-rhombomere boundaries appeared undefined in the Psen1 mutants at E10.5 (data not shown). Rhombomere identity markers indicated that the mass accumulated in r4, but was nevertheless negative for Hoxb1, the r4 marker (Murphy et al., 1989). Its spatial and temporal coordinates suggested that it might correspond to FBM neurons amassing ectopically. In situ hybridization with probes for Phox2b, Tag1 (Cntn2 – Mouse Genome Informatics) and Ret, established markers of migratory FBM neurons(Garel et al., 2000),indicated that FBM neurons were born in ventral r4 of Psen1 mutants,executed their differentiation program by modulating gene expression, but failed nevertheless to engage in caudal migration through r5 and r6(Fig. 1C,D; data not shown). A subset of inner ear efferent (contralateral vestibuloacoustic or CVA) neurons,also born in ventral r4, project axons contralaterally and eventually migrate across the midline (Simon and Lumsden,1993; Bruce et al.,1997). CVA neurons were generated normally in the Psen1mutants, as revealed by Gata3 expression at E11.5(Nardelli et al., 1999; Pata et al., 1999)(Fig. 1E,F), but failed to send axons contralaterally, and presumably to migrate across the midline (see below).

Fig. 1.

Migration of facial branchiomotor neurons and development of the facial nucleus. (A,B) An ectopic mass of postmitotic neurons (arrow in B), revealed on coronal sections by class III β-tubulin in situ hybridization at E11.5, accumulates medially at the level of r4 in the ventral hindbrain of Psen1 mutants (B). (C) At E12.75, Tag1 is expressed in FBM neurons migrating in r4/5/6 in wild-type embryos; (D) FBM fail to migrate in the mutant; bilateral Tag1 expression domains border the ectopic neuronal mass (arrow), visible medially on flat mount of the hindbrain (ventricular view) under Nomarski optics. (E,F) Progenitors of CVA neurons (arrowheads),identified by Gata3 expression at E11.5, are generated normally close to the floor plate in a ventral stripe in r4 in wild-type (E) and mutant (F).(G,H) Ventricular views of flat mounted hindbrains. Retrograde tracing of FBM(bm) and VM (vm) neurons by DiI injection into the root of the VIIth nerve at E11.5. In the wild type (G), the normal trajectory of FBM and VM, normal contralateral projections of the CVA neurons (G, inset) are shown; in the mutant (H), the FBM neurons are stalled. Focal injections of DiO into the ectopic mass (outlined in H after observation under Nomarski optics) only label axons extending unilaterally; CVA contralateral projections are missing from the mutant. Black and white asterisks indicate, respectively, the DiI and DiO injection sites. (I,J) The facial nucleus, detected by Isl1expression on coronal sections at E16.5 is significantly smaller and fragmented in the mutant (J). Notice ectopic Isl1-expressing cells in the floor plate (arrowhead in J). (K,L) Pax6 expression is downregulated in the mutant hindbrain at E11.5 (L). Notice abnormal streams of Pax6-expressing cells in ventral r3 and r5/6 (arrowheads) and the neuronal mass (white arrow). Ventricular view; flat-mount preparations of the ventral hindbrain. r, rhombomere.

Fig. 1.

Migration of facial branchiomotor neurons and development of the facial nucleus. (A,B) An ectopic mass of postmitotic neurons (arrow in B), revealed on coronal sections by class III β-tubulin in situ hybridization at E11.5, accumulates medially at the level of r4 in the ventral hindbrain of Psen1 mutants (B). (C) At E12.75, Tag1 is expressed in FBM neurons migrating in r4/5/6 in wild-type embryos; (D) FBM fail to migrate in the mutant; bilateral Tag1 expression domains border the ectopic neuronal mass (arrow), visible medially on flat mount of the hindbrain (ventricular view) under Nomarski optics. (E,F) Progenitors of CVA neurons (arrowheads),identified by Gata3 expression at E11.5, are generated normally close to the floor plate in a ventral stripe in r4 in wild-type (E) and mutant (F).(G,H) Ventricular views of flat mounted hindbrains. Retrograde tracing of FBM(bm) and VM (vm) neurons by DiI injection into the root of the VIIth nerve at E11.5. In the wild type (G), the normal trajectory of FBM and VM, normal contralateral projections of the CVA neurons (G, inset) are shown; in the mutant (H), the FBM neurons are stalled. Focal injections of DiO into the ectopic mass (outlined in H after observation under Nomarski optics) only label axons extending unilaterally; CVA contralateral projections are missing from the mutant. Black and white asterisks indicate, respectively, the DiI and DiO injection sites. (I,J) The facial nucleus, detected by Isl1expression on coronal sections at E16.5 is significantly smaller and fragmented in the mutant (J). Notice ectopic Isl1-expressing cells in the floor plate (arrowhead in J). (K,L) Pax6 expression is downregulated in the mutant hindbrain at E11.5 (L). Notice abnormal streams of Pax6-expressing cells in ventral r3 and r5/6 (arrowheads) and the neuronal mass (white arrow). Ventricular view; flat-mount preparations of the ventral hindbrain. r, rhombomere.

To corroborate these findings, we injected DiI into the common facial/vestibuloacoustic nerve root at E11.5, in order to trace migrating neurons. In wild-type embryos, a cluster of FBM neurons migrates tangentially along the floor plate in r5 and r6, and visceral motoneurons (VMN) migrate dorsally within r5 (Fig. 1G). Observation at a different focal plane revealed CVA neurons projecting contralaterally across the floor plate(Fig. 1G, inset). In Psen1 mutants, FBM neurons failed to initiate tangential migration and, as a result, accumulated medially at the most distal extent of the leading processes that appeared punctuated. Moreover, CVA neurons did not project contralaterally. Small focal injections of DiO into the ectopic mass labeled axons extending only ipsilaterally towards the exit point of the VIIth nerve (Fig. 1H).

Despite the defect documented at the onset of FBM neuron migration, a small fragmented facial (VII) nucleus was eventually formed, as demonstrated by in situ hybridization with Islet1(Ericson et al., 1992) at E16.5 (Fig. 1I,J), indicating that at least a subset of FBM neurons complete their migratory routine. Therefore, in the absence of Psen1, FBM neurons differentiate, but fail to migrate properly. This defect thus reflects an involvement of Psen1 in tangential neuronal migration.

Further potential migration defects were noted, but studied in less detail. Two other hindbrain somatic motor nuclei were abnormal: the nucleus abducens(VI), differentiating within r5/6, was fragmented; while the hypoglossal (XII)nucleus appeared fused in an aberrant dorsomedial position in r8. Extreme disorganization of hindbrain motoneurons was already obvious at E11.5,evidenced by Isl1 expression (data not shown). Finally, Pax6expression in neuronal progenitors was downregulated in the hindbrain of Psen1 mutants at E11.5 (Fig. 1K,L). Unexpectedly, Pax6-positive cells formed discrete streams in ventral r3 and r5/6 (arrows in Fig. 1L). Taken together, these data suggested that the null mutation in Psen1 affected the migration of somatic motoneurons in the hindbrain. These observations correlate well with the strong expression of Psen1 in most hindbrain nuclei and the facial nucleus in particular (Tanimukai et al., 1999) (A.L., unpublished).

Defects in glia-guided radial migration of cortical neurons

In light of our observations and reports that have indicated cortical dysplasia with focal heterotopias in three independently generated Psen1 strains (Hartmann et al.,1999; Handler et al.,2000; Yuasa et al.,2002), we analyzed cortical development, as an example of radial migration. Psen1 mutants are smaller than wild type, with abnormal overall brain morphology, in part because of severe CNS hemorrhage (Wong et al., 1996; Shen et al., 1996). Because they die perinatally, our analyses were limited to late embryonic stages. Cortical stratification was perturbed in the Psen1 mutants at E16.5, as evidenced in Nissl preparations(Fig. 2A-D). Psen1itself is expressed in wild-type cortex in the ventricular zone (VZ), the intermediate zone (IZ) where cells migrate, the subplate (SP) and the cortical plate (CP) at E17.5 (Fig. 2E,F). Neuronal differentiation, assessed by class IIIβ-tubulin gene expression, appeared to have proceeded at comparable levels in wild-type and Psen1 mutant littermates at late stages (data not shown). Indeed, neuronal differentiation occurs prematurely (and in a region-specific manner) at early stages in Psen1 mutants, but reverts to wild-type rates after E12.5 (Handler et al., 2000).

Fig. 2.

Disorganized cerebral cortex in the Psen1 mutant. (A-D)Nissl-stained coronal sections through wild-type (A,C) and Psen1mutant (B,D) embryos at E16.5 (C and D are higher magnifications of A and B). The ventricular zone, subventricular zone, intermediate zone and cortical plate appear disorganized in the mutant. (E,F) Psen1 is expressed in the ventricular zone, the intermediate zone where cells migrate, the subplate and the cortical plate in E17.5 wild-type embryos. (F) A higher magnification of the area indicated by a black box in E. Sagittal sections; anterior is towards the left. (G,H) BrdU incorporation at E17.5 reveals dense compact bands of BrdU-positive cells in the wild-type (G) and their abnormal patchy distribution in the mutant (H), where bands are disrupted (arrowheads in G,H indicate the ventricular zone). Coronal sections. (I-L) Cortical marker Scip, detected by in situ hybridization on coronal sections of E17.5 embryos, is expressed in the subventricular zone and cortical plate in the wild-type (I,K) but reveals many neurons at ectopic positions in intermediate zone of the mutant (J,L). (K,L) Magnified views of I,J. vz, ventricular zone;svz, subventricular zone; iz, intermediate zone; sp, subplate; cp, cortical plate; mz, marginal zone.

Fig. 2.

Disorganized cerebral cortex in the Psen1 mutant. (A-D)Nissl-stained coronal sections through wild-type (A,C) and Psen1mutant (B,D) embryos at E16.5 (C and D are higher magnifications of A and B). The ventricular zone, subventricular zone, intermediate zone and cortical plate appear disorganized in the mutant. (E,F) Psen1 is expressed in the ventricular zone, the intermediate zone where cells migrate, the subplate and the cortical plate in E17.5 wild-type embryos. (F) A higher magnification of the area indicated by a black box in E. Sagittal sections; anterior is towards the left. (G,H) BrdU incorporation at E17.5 reveals dense compact bands of BrdU-positive cells in the wild-type (G) and their abnormal patchy distribution in the mutant (H), where bands are disrupted (arrowheads in G,H indicate the ventricular zone). Coronal sections. (I-L) Cortical marker Scip, detected by in situ hybridization on coronal sections of E17.5 embryos, is expressed in the subventricular zone and cortical plate in the wild-type (I,K) but reveals many neurons at ectopic positions in intermediate zone of the mutant (J,L). (K,L) Magnified views of I,J. vz, ventricular zone;svz, subventricular zone; iz, intermediate zone; sp, subplate; cp, cortical plate; mz, marginal zone.

In cerebral cortex, radial glial cells are thought both to generate neurons and guide their radial migration (Noctor et al., 2001). A single-dose BrdU pulse at E17.5 labeled the nuclei of dividing cells – presumed to be radial glia – in the VZ. Labeled cells formed an orderly, thin band along the ventricular surface of wild-type cortex (Fig. 2G) but in the mutant, patches of dense labeled nuclei alternated with patches of sparse labeling (Fig. 2H),suggesting that radial glia progenitor cells were abnormally positioned or dysfunctional. Previous studies have documented only mild and localized differences in BrdU labeling patterns in the Psen1 mutants at early stages (Handler et al.,2000).

To assess migration to and within the cortical plate at E17.5, we used a panel of layer-specific markers for genes [reelin, Scip (Pou3f1– Mouse Genome Informatics), Tbr1 and p75(Ngfr – Mouse Genome Informatics)]. The earliest generated marginal zone (MZ), which is identified by reelin expression, formed normally in the Psen1 mutants, indicating that migration of Cajal-Retzius cells was unaffected. The layer of Cajal-Retzius cells appeared nevertheless disrupted, perhaps owing to overall reduction of cell density in the MZ(Hartmann et al., 1999). Expression of Scip is confined to a subpopulation of prospective layer V neurons, but is also high in the subventricular (SVZ) and intermediate(IZ) zones at late embryonic stages(Frantz et al., 1994). In Psen1 mutants, Scip expression was indeed detected within the CP; strikingly, however, many neurons expressed Scip ectopically along a radial path, as though unable to execute their migratory program properly (Fig. 2I-L). At late embryonic stages, Tbr1 is expressed in the subplate and future layer VI, as well as in superficial layers I-III(Bulfone et al., 1995). Expression of Tbr1 in the Psen1 mutants again revealed apparent abnormal migratory behavior of differentiating cortical neurons(Fig. 3A,B). Expression of p75, which was confined to the subplate and layer VI at this stage(Mackarehtschian et al.,1999), was not only downregulated in the Psen1 mutants,but also appeared patchy, lacking normal gradients, and severely disorganized(Fig. 3C,D). Furthermore,expression of cadherin 6 and cadherin 8, which were detected, respectively, in future layers II-IV and V/VI (Inoue et al., 1998), was downregulated overall in the CP and nearly absent in the IZ of Psen1 mutants (Fig. 3E,F; data not shown).

Fig. 3.

Disruption of cortical laminar and radial glial markers. (A,B) Tbr1, expressed in the subplate and upper cortical plate in the wild-type (A), indicates abnormal migration of cortical neurons in the Psen1 mutant (B). (C,D) p75, which is detected in the subplate and future layer VI in the wild type (C), is downregulated in the mutant and nearly absent from the ventricular zone (D). (E,F) Cdh6expression labels neurons more sparsely in the mutant (F) than in wild type(E), particularly in the ventricular and intermediate zone (F). (G-J) Radial glia markers in the ventricular zone are severely downregulated in the mutant(H,J) in comparison with wild type (G,I). (G,H) Dab1. (I,J) Pax6. (K,L) Notch pathway activity, visualized by Hes5expression in the wild type (K) is dramatically reduced in the mutant (L). In situ hybridization on coronal sections through E17.5 (A-F) or E16.5 (G-L)cortex. vz, ventricular zone.

Fig. 3.

Disruption of cortical laminar and radial glial markers. (A,B) Tbr1, expressed in the subplate and upper cortical plate in the wild-type (A), indicates abnormal migration of cortical neurons in the Psen1 mutant (B). (C,D) p75, which is detected in the subplate and future layer VI in the wild type (C), is downregulated in the mutant and nearly absent from the ventricular zone (D). (E,F) Cdh6expression labels neurons more sparsely in the mutant (F) than in wild type(E), particularly in the ventricular and intermediate zone (F). (G-J) Radial glia markers in the ventricular zone are severely downregulated in the mutant(H,J) in comparison with wild type (G,I). (G,H) Dab1. (I,J) Pax6. (K,L) Notch pathway activity, visualized by Hes5expression in the wild type (K) is dramatically reduced in the mutant (L). In situ hybridization on coronal sections through E17.5 (A-F) or E16.5 (G-L)cortex. vz, ventricular zone.

A common denominator in gene expression pattern changes described above was lower than normal intensity in the VZ of the Psen1 mutants(Fig. 2I-L; Fig. 3A-F). We analyzed expression of radial glial markers, on one hand, and of Dab1, on the other, as components of the reelin pathway are expressed in the VZ(Tissir and Goffinet, 2003). Dab1 was downregulated in the CP of Psen1 mutants at E16.5,and nearly undetectable in the VZ (Fig. 3G,H). Interestingly, Dab1 has recently been shown to colocalize with radial glial markers in the VZ(Luque et al., 2003), and to influence neuronal migration by controlling the timing of detachment of migrating neurons from radial glia (Sanada et al., 2004). Pax6, which is localized in radial glia in the E16 cortex (Götz et al.,1998), was severely downregulated in the Psen1 mutants(Fig. 3I,J). Finally, the dramatic downregulation of Hes5, a target of the Notch pathway in neural progenitors and a faithful read-out of its activity(Ahmad et al., 1995; Ohtsuka et al., 1999),indicated that Notch signaling was suboptimal in the Psen1 mutants(Fig. 3K,L). This result is consistent with findings described above, in that activation of Notch signaling promotes context-dependent radial glial fate(Gaiano et al., 2000).

The molecular data described above, in conjunction with the abnormal distribution of BrdU at E17.5, pointed, albeit indirectly, to a defect in radial glia. To directly examine its morphology, we labeled pia-attached radial glia by placing small DiI crystals along the pial surface of the brain at E16.5. In wild type, radial glia processes were rather straight and regular, resembling well-combed hair, and extended to the ventricular surface. By contrast, in the Psen1 mutant, radial glial processes appeared tangled (Fig. 4A-D). Morphological changes in radial glia were confirmed by RC2 immunohistochemistry (Fig. 4E,F). To assess the association of neurons with radial glia, DiI was photoconverted and sections were processed to show Scipexpression. In wild-type cortex, Scip-expressing neurons were smoothly juxtaposed to radial glial processes(Fig. 4G). In the mutant, thick masses of Scip-expressing cells and their processes overlaid clumps of radial glia, suggesting stalled or defective migration(Fig. 4H). Both molecular and morphological observations therefore pointed to defects in cortical proliferation and radial migration, at least in part due to abnormalities in radial glial cells.

Fig. 4.

Radial glia abnormalities. (A-D) Radial glia are labeled by DiI injection in the pial surface of E16.5 cortex, sectioned in the coronal plane (100μm) after diffusion of the dye. In wild type, radial glia fibers are regular and labeled in their entire length from the pial to the ventricular surface (A,C), but appear tangled and often fail to label all the way to the ventricular surface in the Psen1 mutant (B,D). Two examples of aggregated fibers are shown in D. (E,F) Radial glial morphology is revealed by RC2 immunofluorescence on horizontal sections through E14.5 cortex. Radial glial processes are smooth and long in the wild-type (E, arrowheads), but irregular in the mutant (F). Notice intense staining towards the radial glial end-feet. Scale bar: 50 μm. (G,H) DiI was photoconverted on sections equivalent to those shown in (A,B) and Scip-expressing cells were detected by in situ hybridization. In wild type (G), glial fibers (brown) are regular. Black arrows indicate Scip-positive neurons (blue), which are also DiI labeled, presumably because the dye spread into the neurons from the radial glia to which they are attached. In the mutant (H), radial glial fibers aggregate and run at angles to one another (black arrow). Scip-positive cells (blue) are massed within the radial glial aggregates. White arrow in H indicates several Scip-positive cell bodies. All sections are shown with pial side upwards.

Fig. 4.

Radial glia abnormalities. (A-D) Radial glia are labeled by DiI injection in the pial surface of E16.5 cortex, sectioned in the coronal plane (100μm) after diffusion of the dye. In wild type, radial glia fibers are regular and labeled in their entire length from the pial to the ventricular surface (A,C), but appear tangled and often fail to label all the way to the ventricular surface in the Psen1 mutant (B,D). Two examples of aggregated fibers are shown in D. (E,F) Radial glial morphology is revealed by RC2 immunofluorescence on horizontal sections through E14.5 cortex. Radial glial processes are smooth and long in the wild-type (E, arrowheads), but irregular in the mutant (F). Notice intense staining towards the radial glial end-feet. Scale bar: 50 μm. (G,H) DiI was photoconverted on sections equivalent to those shown in (A,B) and Scip-expressing cells were detected by in situ hybridization. In wild type (G), glial fibers (brown) are regular. Black arrows indicate Scip-positive neurons (blue), which are also DiI labeled, presumably because the dye spread into the neurons from the radial glia to which they are attached. In the mutant (H), radial glial fibers aggregate and run at angles to one another (black arrow). Scip-positive cells (blue) are massed within the radial glial aggregates. White arrow in H indicates several Scip-positive cell bodies. All sections are shown with pial side upwards.

Corticogenesis also depends on the tangential migration of some cell types,notably of interneurons from the ganglionic eminences. Expression of Dlx2 (Porteus et al.,1994) identified in wild-type embryos a well-developed region adjacent to the ganglionic eminences (Fig. 5A), from which a faint stream of cells appeared to be migrating into the cortex through the SVZ (arrow in Fig. 5A). In Psen1mutants, however, the Dlx2-positive region was abnormal(Fig. 5B), with more migrating cells evident (arrow in Fig. 5B). Expression of glutamic acid decarboxylase (Gad67; Gad1 – Mouse Genome Informatics) revealed that Gad67-positive cells, normally engaging in tangential migration through deep layers, had instead aggregated superficially(Fig. 5C,D; arrow in Fig. 5D).

Fig. 5.

Tangential migrations in the cortex. (A,B) Dlx2-expressing zone in the wild type (A) (high magnification in a) from which cells migrate into the cortex (arrowhead in A). In the Psen1 mutant (B), the Dlx2-positive domain (high magnification in b) is abnormal, and migration into the cortex appears enhanced (arrowhead in B). (C,D) Gad67-positive cells engage in tangential migration through deep layers in the wild-type (C), but aggregate superficially in the mutant (arrow in D). Ventral quadrant view of coronal sections through E17.5 embryos processed for in situ hybridization.

Fig. 5.

Tangential migrations in the cortex. (A,B) Dlx2-expressing zone in the wild type (A) (high magnification in a) from which cells migrate into the cortex (arrowhead in A). In the Psen1 mutant (B), the Dlx2-positive domain (high magnification in b) is abnormal, and migration into the cortex appears enhanced (arrowhead in B). (C,D) Gad67-positive cells engage in tangential migration through deep layers in the wild-type (C), but aggregate superficially in the mutant (arrow in D). Ventral quadrant view of coronal sections through E17.5 embryos processed for in situ hybridization.

Finally, the dentate gyrus, another site of tangential migration, appeared poorly formed as assessed by NeuroD (Neurod1 – Mouse Genome Informatics) and Prox1 expression (data not shown). Taken together,our observations suggest that cortical plate formation, which relies on radial migration, and tangential migration of interneurons from the basal telencephalon into the cortex were both affected in the absence of Psen1.

Migration defects in the midbrain

We next analyzed the effects of Psen1 loss of function in the midbrain,where neuronal migration has not been studied extensively. We noticed that expression of F-spondin (Spon1 – Mouse Genome Informatics), which is implicated in spinal cord commissural axon pathfinding(Burstyn-Cohen et al., 1999)was severely diminished in the Psen1 mutants at E10.5 and E11.5(Fig. 6A,B; data not shown). However, ventral midbrain expression of other markers (Shh, Lmx1a, Lmx1b,Pax6) between E10.5 and E12.5 appeared normal(Fig. 6C,D; data not shown). Moreover, the anlagen of the oculomotor complex (III) and the trochlear (IV)motor nucleus, identified, respectively, by Isl1/Gata3/Phox2b expression, or Phox2bexpression alone (Pattyn et al.,1997; Nardelli et al.,1999; Agarwala and Ragsdale,2002) formed in the Psen1 mutants(Fig. 6E,F; data not shown);the former, however, was smaller and had fewer Isl1/Gata3-positive cells migrating anteriorly(Fig. 6F).

Fig. 6.

Midbrain development. (A-D) Whole-mount in situ hybridization on E11.5 embryos. Expression of molecular markers in the ventral midbrain in wild type(A,C) and Psen1 mutant (B,D) embryos. (A,B) F-spondin expression is specifically downregulated in the ventral midbrain (and diencephalon), but unaffected in the hindbrain of the mutant. (C,D) By contrast, no differences are detected in the expression pattern of Lmx1a between wild type (C)and mutant (D). (E-H) Anlagen of midbrain nuclei are formed abnormally in the Psen1 mutants. (E,F) Flat-mount preparations of E11.5 embryos,ventricular view; anterior towards the top. The oculomotor complex, which is detected by Gata3 expression, is smaller in the mutant (F) in comparison with wild type (E). (G-J) Midbrain dopaminergic neurons (DA),detected by Th expression at E11.5, develop close to the ventral midline in the wild-type (G) but are continuous across the midline in the mutant (H). (I,J) At late embryonic stages (E17.5), DA neurons remain clustered at the midline of the mutant (J). Notice ectopic cluster of Th-positive neurons (arrow in J). Coronal sections through E11.5(G,H) and E17.5 (I,J) embryos. aq, aqueduct; red arrowheads in H,J indicate the midline.

Fig. 6.

Midbrain development. (A-D) Whole-mount in situ hybridization on E11.5 embryos. Expression of molecular markers in the ventral midbrain in wild type(A,C) and Psen1 mutant (B,D) embryos. (A,B) F-spondin expression is specifically downregulated in the ventral midbrain (and diencephalon), but unaffected in the hindbrain of the mutant. (C,D) By contrast, no differences are detected in the expression pattern of Lmx1a between wild type (C)and mutant (D). (E-H) Anlagen of midbrain nuclei are formed abnormally in the Psen1 mutants. (E,F) Flat-mount preparations of E11.5 embryos,ventricular view; anterior towards the top. The oculomotor complex, which is detected by Gata3 expression, is smaller in the mutant (F) in comparison with wild type (E). (G-J) Midbrain dopaminergic neurons (DA),detected by Th expression at E11.5, develop close to the ventral midline in the wild-type (G) but are continuous across the midline in the mutant (H). (I,J) At late embryonic stages (E17.5), DA neurons remain clustered at the midline of the mutant (J). Notice ectopic cluster of Th-positive neurons (arrow in J). Coronal sections through E11.5(G,H) and E17.5 (I,J) embryos. aq, aqueduct; red arrowheads in H,J indicate the midline.

Midbrain dopaminergic (DA) neurons are born ventrally and thought to migrate extensively to their final positions(Altman and Bayer, 1981). Migrating DA neurons were identified by tyrosine hydroxylase (Th)expression (Specht et al.,1981). DA neurons appeared fused across the midline in the Psen1 mutants at E11.5 (Fig. 6G,H), where Th-expressing cells also persisted later(Fig. 6I,J). Thus, DA neurons appear to represent yet another example of a cell type that differentiates but does not migrate properly, becoming stalled at abnormal positions.

Finally, the two bilateral streams of cells emigrating caudally from the mesencephalic tract of the trigeminal nerve (tmesV) into dorsal r1, identified by Isl1 expression at E11.5(Fedtsova and Turner, 2001),were considerably underdeveloped (data not shown).

Defects in tangential migration in the brainstem

Next, we focused on the development of the cerebellum and precerebellar system for several reasons: the rhombic lip is a site of tangential (posterior to anterior and circumferential) migrations par excellence; Psen1 is expressed in the cerebellum, the pontine nucleus and the inferior olive(Lee et al., 1996; Tanimukai et al., 1999); and,finally, the caudal midbrain appears to have overgrown at the expense of an abnormal cerebellum.

Prenatal cerebellar development relies on tangential migration of the proliferative granule cell precursors (GCPs) from the anterior rhombic lip over the developing cerebellum, and ascension of Purkinje cells (PCs) from the neuroepithelium along radial glia fibers(Altman and Bayer, 1997). In Psen1 mutants, the cerebellum was smaller with its two halves remaining separate posteriorly, indicating incomplete fusion at the midline(Fig. 7A-H). To examine GCPs at E17.5 we analyzed Math1 (Atoh1 – Mouse Genome Informatics) (Ben-Arie et al.,1997) and Pax6(Engelkamp et al., 1999). A well-developed external granule layer (EGL) covered the cerebellum in wild type; in the Psen1 mutants, however, GCPs failed to reach the anterior-most part of the cerebellum (Fig. 7A-B,E-F,G-H), where a GCP-free medial region developed (arrow in Fig. 7B,D). In addition, PCs,which are identified by Rora expression(Hamilton et al., 1996), had clustered beneath the displaced EGL (Fig. 7C,D,I,J; compare 7A with 7C, 7B with 7D, 7G with 7I, and 7H with 7J).

Fig. 7.

Development of the cerebellum and precerebellar nuclei. (A-D) Development of the external granular layer and the Purkinje cells, detected respectively by Math1 and Rora expression in wild type (A,C) and Psen1 mutant (B,D) embryos. (A,B) GCP generation and spreading are compromised in the mutant. A,C and B,D are adjacent, mid-sagittal sections through E17.5 cerebellum. (C,D) Purkinje cells form clusters in the mutant beneath the EGL (D). In the mutant (B,D), there is overgrowth of caudal midbrain (asterisks) and presence of medial tissue (indicated by two white lines and black arrow) devoid of GCPs and PCs. (E-J) Coronal sections through E17.5 cerebellum at the levels (e and g, i) indicated by white lines in A. Midline fusion of posterior cerebellum is incomplete in the mutant. (E-H) In situ hybridization with Pax6 identifies GCPs. Cerebellar morphology is affected medially (F) and posterior cerebellum remains unfused in mutant(asterisk in H) when compared with wild type (E,G). This morphogenetic defect causes GCPs to cluster (H). (I,J) PCs, which are identified by Roraexpression, assume positions beneath the normally developing EGL in wild type(I) and the displaced EGL in the mutant (J). (K,L) Pontine migratory stream and pontine nucleus proper detected by Pax6 expression at E16.5. The migratory stream is underpopulated, Pax6-expressing cells accumulate ectopically (arrow in L) and the pontine nucleus (pn) is severely underdeveloped in the mutant (L). In situ hybridization on sagittal (A-D and K,L) and coronal (E-J) sections through caudal midbrain and cerebellum at E17.5 (A-J) or E16.5 (K,L).

Fig. 7.

Development of the cerebellum and precerebellar nuclei. (A-D) Development of the external granular layer and the Purkinje cells, detected respectively by Math1 and Rora expression in wild type (A,C) and Psen1 mutant (B,D) embryos. (A,B) GCP generation and spreading are compromised in the mutant. A,C and B,D are adjacent, mid-sagittal sections through E17.5 cerebellum. (C,D) Purkinje cells form clusters in the mutant beneath the EGL (D). In the mutant (B,D), there is overgrowth of caudal midbrain (asterisks) and presence of medial tissue (indicated by two white lines and black arrow) devoid of GCPs and PCs. (E-J) Coronal sections through E17.5 cerebellum at the levels (e and g, i) indicated by white lines in A. Midline fusion of posterior cerebellum is incomplete in the mutant. (E-H) In situ hybridization with Pax6 identifies GCPs. Cerebellar morphology is affected medially (F) and posterior cerebellum remains unfused in mutant(asterisk in H) when compared with wild type (E,G). This morphogenetic defect causes GCPs to cluster (H). (I,J) PCs, which are identified by Roraexpression, assume positions beneath the normally developing EGL in wild type(I) and the displaced EGL in the mutant (J). (K,L) Pontine migratory stream and pontine nucleus proper detected by Pax6 expression at E16.5. The migratory stream is underpopulated, Pax6-expressing cells accumulate ectopically (arrow in L) and the pontine nucleus (pn) is severely underdeveloped in the mutant (L). In situ hybridization on sagittal (A-D and K,L) and coronal (E-J) sections through caudal midbrain and cerebellum at E17.5 (A-J) or E16.5 (K,L).

As noted further below, these defects in cerebellar morphogenesis are likely to represent the combinatorial outcome of abnormal proliferation as well as differentiation and migration.

Development of the precerebellar system, however, relies on tangential migrations from the posterior rhombic lip(Altman and Bayer, 1997; Rodriguez and Dymecki, 2000). For example, pontine nuclei are formed by long-range migration of postmitotic precursors along a superficial circumferential trajectory. We used Pax6 as a marker of the pontine migratory stream and nuclei proper(Engelkamp et al., 1999; Yee et al., 1999). Pontine nuclei were underdeveloped in the Psen1 mutants, as evidenced by morphology and Pax6 expression and the migratory stream was underpopulated with migratory precursors, only a few of which appeared to reach their final destination in the pons(Fig. 7K,L). Instead, a cluster of Pax6-positive cells accumulated ectopically(Fig. 7L, arrow). Finally, the inferior olive appeared poorly assembled (data not shown). Thus, the pontine nuclei appear to represent another example of a tangential migration defect in the absence of Psen1.

Defects in morphogenesis of the mid/hindbrain

Gross morphological and gene expression analyses indicated defects in the derivatives of the mid/hindbrain (see Fig. 7A-D). A single-dose BrdU pulse at E17.5 identified the compromised EGL in the Psen1 mutants(Fig. 8A,B), and numerous proliferating cells in the GCP-free ectopic region rostral to the vestigial cerebellum, as well as in the caudal midbrain(Fig. 8B). Reelin expression confirmed that the EGL was abnormal, and emphasized the dramatic overgrowth of the inferior colliculus (the caudal part of dorsal midbrain), normally thinner than the superior colliculus (the rostral part) at this stage(Fig. 8C,D; asterisk in D). The expression domain of ephrin A5 (Donoghue et al., 1996) was dramatically enlarged, indicating that the inferior colliculus was indeed expanded in the Psen1 mutants(Fig. 8E,F).

Fig. 8.

Morphogenetic defects in the mid/hindbrain region. (A,B) BrdU incorporation at E17.5 detects proliferating cells in the EGL of wild type (A) and Psen1 mutants (B). In the mutant, proliferation is highest in medial ectopic tissue (white arrow in B) and also in the caudal midbrain (black arrow in B). (C,D) Expression of reelin is detected in the EGL of wild type (C) and mutant (D), and reveals, in addition, a dramatic overgrowth of the caudal midbrain in the mutant (asterisk in D) and a medial mass forming between the developing cerebellum and the caudal midbrain (delineated by the two white lines in D). (E,F) Ephrin A5 expression identifies the inferior colliculus in wild type (E) and its expansion in the mutant (F). Sagittal (A-D) or coronal(E,F) sections through caudal midbrain and cerebellum at E17.5.

Fig. 8.

Morphogenetic defects in the mid/hindbrain region. (A,B) BrdU incorporation at E17.5 detects proliferating cells in the EGL of wild type (A) and Psen1 mutants (B). In the mutant, proliferation is highest in medial ectopic tissue (white arrow in B) and also in the caudal midbrain (black arrow in B). (C,D) Expression of reelin is detected in the EGL of wild type (C) and mutant (D), and reveals, in addition, a dramatic overgrowth of the caudal midbrain in the mutant (asterisk in D) and a medial mass forming between the developing cerebellum and the caudal midbrain (delineated by the two white lines in D). (E,F) Ephrin A5 expression identifies the inferior colliculus in wild type (E) and its expansion in the mutant (F). Sagittal (A-D) or coronal(E,F) sections through caudal midbrain and cerebellum at E17.5.

To assess early stages of cerebellar morphogenesis, we analyzed Math1 and Pax6 expression. Expression of Math1 was normal at E10.5 but became significantly enhanced in the mutants at E11.5 and persisted in the medial rhombic lip, where it is normally downregulated(Louvi et al., 2003). At E14.5, the rhombic lip appeared thinned and Math1-expressing cells accumulated in a broad medial domain, failing to spread anterodorsally; in addition, the overall morphology of the developing cerebellum was abnormal(Fig. 9A,B). In parallel, we noticed a delay in the onset of Pax6 expression in the rhombic lip(data not shown). We analyzed Otx2 and Gbx2, which are involved in the positioning and maintenance of the isthmic organizer (reviewed by Wurst and Bally-Cuif,2001): whereas Otx2 appeared unaffected at E11.5 and E12.5, Gbx2 was downregulated in r1 in particular (and anterior hindbrain in general), indicating that this important signaling center was possibly deregulated (Fig. 9C-F). Finally, precursors of the deep cerebellar nuclei appeared to be generated normally (data not shown). These data implied that, on one hand, proliferation and differentiation of granule cell precursors were probably both affected, interfering with correct execution of migration; and,on the other, the isthmic organizer was deregulated, ultimately leading to abnormal morphogenesis of the mid/hindbrain region and its derivatives.

Fig. 9.

Early development of the cerebellum. (A,B) Incorrect regulation of Math1 expression in the anterior rhombic lip. At E11.5, Math1 expression is enhanced in the Psen1 mutants (B, upper panel) in comparison with wild type (A, upper panel). At E13.5, strong Math1 expression persists in the medial rhombic lip of the mutant(arrow in B, middle panel) but becomes downregulated in the wild type (A,middle panel). At E14.5, Math1-positive cells are spreading over the cerebellum in the wild type (A, lower panel), but accumulate medially in the mutant (B, lower panel). Notice the thinned rhombic lip and overall abnormal morphology of cerebellum in the mutant. (C-F) Downregulation of Gbx2expression in anterior hindbrain. (C,D) Gbx2 expression is downregulated in the mutant. Dorsal view of wild type (C) and mutant (D)embryos after whole-mount in situ hybridization. Gbx2 expression is lower in dorsal r1 of the mutant (arrow in D). (E,F) Flat-mount preparations of hindbrain following in situ hybridization with Gbx2. Notice downregulation and incorrect pattern of expression (asterisks in F) in the mutant anterior hindbrain.

Fig. 9.

Early development of the cerebellum. (A,B) Incorrect regulation of Math1 expression in the anterior rhombic lip. At E11.5, Math1 expression is enhanced in the Psen1 mutants (B, upper panel) in comparison with wild type (A, upper panel). At E13.5, strong Math1 expression persists in the medial rhombic lip of the mutant(arrow in B, middle panel) but becomes downregulated in the wild type (A,middle panel). At E14.5, Math1-positive cells are spreading over the cerebellum in the wild type (A, lower panel), but accumulate medially in the mutant (B, lower panel). Notice the thinned rhombic lip and overall abnormal morphology of cerebellum in the mutant. (C-F) Downregulation of Gbx2expression in anterior hindbrain. (C,D) Gbx2 expression is downregulated in the mutant. Dorsal view of wild type (C) and mutant (D)embryos after whole-mount in situ hybridization. Gbx2 expression is lower in dorsal r1 of the mutant (arrow in D). (E,F) Flat-mount preparations of hindbrain following in situ hybridization with Gbx2. Notice downregulation and incorrect pattern of expression (asterisks in F) in the mutant anterior hindbrain.

Loss of Psen1 function results in multiple abnormalities in brain structure at the end of embryogenesis. We have documented widespread, albeit not universal, defects in neuronal migration and CNS morphogenesis in the Psen1 mutants affecting both radial as well as tangential migration and leading to abnormal morphogenesis of the cerebral cortex and the brainstem. The sites where these Psen1-associated defects are seen correlate well with regions of high levels of Psen1 mRNA expression, consistent with the notion that Psen1 may indeed be implicated in these processes, but also suggesting a far broader role of Psen1 in brain development than previously assumed. Importantly, our analysis indicates that the phenotype we document is likely to be the consequence of abnormalities in a diverse set of cellular pathways that are linked directly or indirectly to Psen1 function.

Psen1 and Cdk5 mutants: similarities and implications

The array of neuronal migration defects in the Psen1 mutants is strikingly reminiscent of those observed in embryos with targeted mutations in the Cdk5/p35/p39 pathway (reviewed by Dhavan and Tsai, 2001; Ohshima and Mikoshiba, 2002). In the cerebral cortex, Cdk5 mutants exhibit defects confined to the late-migrating, glia-guided cortical neurons. Cdk5 and Psen1mutants share a surprisingly similar defect in the positioning of FBM,complete with the appearance of an ectopic mass of postmitotic neurons in the ventral hindbrain. Dhavan and Tsai (Dhavan and Tsai, 2001) have previously suggested that Cdk5 and its regulatory subunits p35 and p39 are crucial regulators of neuronal migration. Indeed, p35 mutants display an atypical mode of migration in the cortex, associated with disrupted neuronal-glial interactions(Gupta et al., 2003). The similarities in the mutant phenotypes we document indicate the possible convergence of different pathways towards similar intracellular factors and could be the manifestation of molecular interactions between the Psen1 and Cdk5 ‘pathways’. Psen1 has been reported to interact with Cdk5 in many ways. On the one hand, Cdk5/p35 has been reported to bind and phosphorylate β-catenin and to regulate β-catenin/Psen1 interactions(Kesavapany et al., 2001). Cdk5/p35 is, on the other hand, involved in the regulation of N-cadherin-mediated adhesion in cortical neurons, and N-cadherin itself is aγ-secretase substrate (Kwon et al.,2000; Marambaud et al.,2002). Dab1, which is downregulated in Psen1 mutants, is also a substrate for Cdk5/p35, and interacts with APP in yeast two-hybrid screens (Howell et al., 1999; Keshvara et al., 2002). Finally, deregulation of Cdk5 activity through accumulation of p25 (a cleavage product of p35) has been implicated in Alzheimer’s disease itself(Tseng et al., 2002).

In comparing Psen1-related neuronal migration phenotypes to the phenotypic consequences of other mutations, it is noteworthy that recent observations suggest an intersection of pathways controlling molecular mechanisms of neuronal migration and axonal transport. In legs at odd angles(Loa/Loa) embryos, which carry a missense mutation in dynein cytoplasmic heavy chain, migrating FBM neurons bifurcate and a double nucleus eventually forms (Hafezparast et al.,2003), not unlike the fragmented facial nucleus of the Psen1 mutants. Cytoplasmic dynein is a major motor complex involved in retrograde transport with reported roles in neuronal migration, neurite outgrowth and axonal transport of microtubules, neurofilaments and organelles(reviewed by Morris, 2000; Terada and Hirokawa, 2000). Interestingly, the dynein pathway component Nudel is a substrate for Cdk5(Niethammer et al., 2000; Sasaki et al., 2000),suggesting a further link. Moreover, embryos lacking another motor protein,kinesin KIF1Bβ, also display defective development of the facial nucleus(Zhao et al., 2001). Notably,Psen1 has recently been implicated in anterograde (kinesin-based) axonal transport (Pigino et al.,2003).

Psen1 and Small eye mutants: the role of Pax6

It is quite clear that the abnormalities we observe in the Psen1mutants cannot be explained only by the Psen1/Cdk5 relationship. Our analysis revealed a second process that appears to be affected by Psen1 malfunction,namely Pax6 regulation. We showed that, in the Psen1 mutants in general, Pax6 expression is downregulated (in the VZ of the developing cortex, the hindbrain and the precerebellar nuclei) or delayed (in the rhombic lip), and is relatively unaffected only in the ventral midbrain at early stages. The Pax6 expression changes we observe offer a plausible explanation for part of the phenotypes we documented. Hints of a possible relationship between Pax6 and Psen1 come from the wealth of information available on the small eye (Sey) mouse, which lacks functional Pax6 protein (Hill et al.,1991). In the Sey/Sey developing cortex neuronal migration defects occur primarily at late stages, indicating that they might be radial glia-dependent (Schmahl et al.,1993; Caric et al.,1997). Indeed, radial glia are lost in the Sey/Sey cortex(Götz et al., 1998). Moreover, in the Sey/Sey mouse, loss of Pax6 leads to excessive migration of interneurons from basal brain into the cortex(Chapouton et al., 1999; Yun et al., 2001), hindbrain motor neurons are incorrectly specified(Ericson et al., 1997; Osumi et al., 1997) and rhombic lip-derived structures severely affected(Engelkamp et al., 1999). Widespread downregulation of Pax6 expression as such documented in the Psen1 mutants is therefore expected to affect much the same processes. Thus, the defects in radial-glia dependent cortical migration,radial glia differentiation in the VZ, incorrect migration of interneurons into the cortex and of pontine precursors in the precerebellar system could be consequences of insufficient levels of Pax6 expression. Although no direct relationship between Pax6 and Psen1 has been described, studies in Drosophila have linked Notch signaling with the regulation of the Pax6 ortholog eyless (ey) during eye development,suggesting that ey may act, in some fashion, downstream of Notch signal input (Kurata et al.,2000; Kumar and Moses,2001; Kenyon et al.,2003). One possibility, therefore, is that Psen1 influences Pax6 function via the Notch pathway.

Morphogenesis of the cerebellar system, tangential migrations and guidance molecules

We found that loss of Psen1 function leads to abnormalities in glia-guided radial migration, as well as tangential migration, and to defects in cerebellar morphogenesis. We determined that the latter is accompanied by the downregulation of Gbx2 in the anterior hindbrain. Otx2 and Gbx2 act antagonistically to position the isthmic organizer (reviewed by Wurst and Bally-Cuif, 2001),and although Otx2 appears unaffected in the Psen1 mutants, Gbx2 downregulation implies that the isthmic organizer might be deregulated. Interestingly, in the absence of functional Gbx2, the inferior colliculus is dramatically thickened(Wassarman et al., 1997), a phenotype also observed in the Psen1 mutants. In addition to Gbx2 downregulation, incorrect regulation of Math1 may partly account for the cerebellar morphogenetic phenotype: downregulation of Math1 in the medial rhombic lip is essential for cerebellar midline fusion (Louvi et al., 2003). What could account for the failure of CGPs to reach the anterior-most part of the cerebellum? Although we have not examined this issue in detail, we note that the guidance molecule Erbb4, subject to γ-secretase processing(Ni et al., 2001), is expressed in GCPs and in the caudal rhombic lip(Dixon and Lumsden, 1999),explaining perhaps part of the migration defects we observe. In addition, it is known that precerebellar nuclei precursors interpret netrin as a chemoattractant (reviewed by Wingate,2001) and this property could account for the defects in the pontine migratory stream and nuclei. Interestingly, of all netrin receptors,the migratory pontine cells express on their leading processes solely DCC(Yee et al., 1999) and not surprisingly, pontine nuclei are absent in netrin 1 and Dcc mutant mice (Serafini et al., 1996; Fazeli et al., 1997). Moreover, cells migrating to the inferior olive express Dcc (in addition to other netrin receptors) and netrin signaling plays a role in the finer subdivision of this nucleus. Although netrin expression appears unaffected in the ventral hindbrain of Psen1 mutants, Dcc was recently shown to undergo Psen1-dependent γ-secretase processing(Taniguchi et al., 2003),suggesting that the interpretation and/or reception of netrin signaling is defective in the absence of functional Psen1.

Presenilins and the cytoskeleton: a mechanistic explanation?

As radial and tangential modes of migration are affected in the Psen1 mutants, it seems plausible that fundamental cellular mechanisms required for cell movement might be perturbed in the absence of functional Psen1 protein. In preparation to move, cells extend a leading process sensing the immediate environment, followed by translocation of the nucleus into the leading process and subsequent retraction of the trailing process. The first step heavily depends on polymerization and reorganization of actin microfilaments and is controlled by Rho family GTPases(Ridley, 2001), while the second step relies on microtubules (Morris et al., 1998; Lambert de Rouvroit and Goffinet, 2001; Nadarajah and Parnavelas,2002). Evidence suggests that Psen1 may indeed interact with cytoskeletal elements. First, in hippocampal cultures, Psen1 associates with microtubules and microfilaments in a developmentally regulated manner and is localized in lamellipodia and filopodia of neuronal growth cones(Pigino et al., 2001). Second,the microtubule-associated protein Tau can associate with Psen1 in cultured cells and to a lesser extent in brain extracts(Takashima et al., 1998). Third, presenilins can interact in vivo and in vitro with at least two actin-binding family members, filamin A and filamin homolog 1(Zhang et al., 1998). In Drosophila, filamin interacts genetically and physically with presenilin (Guo et al., 2000). More importantly, in humans, mutations in filamin A prevent migration of cerebral cortical neurons causing periventricular heterotopia(Fox et al., 1998). Finally,in Drosophila, Psn (presenilin) mutations disrupt the spectrin cytoskeleton (López-Schier and St Johnston, 2002), whereas Psen1-dependent γ-secretase cleavage of E-cadherin leads to its dissociation from the cytoskeleton(Marambaud et al., 2002). Interestingly enough, α-spectrin accumulates in cytoplasmic inclusions in the brains of individuals with Alzheimer’s disease(Sangerman et al., 2001). Thus, evidence, albeit circumstantial, exists to suggest a link between presenilins and the cytoskeleton that could provide a mechanistic explanation for some of the migration defects seen in the Psen1 mutants.

In conclusion, our analysis established a novel role for Psen1 in neuronal migration and morphogenesis while revealing the complex relationship between Psen1 and specific cellular events and biochemical pathways that affect migration. The extent to which this complex phenotype directly or indirectly reflects the diversity of substrates that can be affected by γ-secretase remains to be determined. Nevertheless, our data implicate Psen1 with a roster of specific cellular pathways and demonstrate that the Psen1 loss-of-function phenotypes reflect the many developmental processes simultaneously affected by this mutation.

We are grateful to Kamal Sharma for his hospitality and support. We thank Michio Yoshida and Marion Wassef for discussion and comments on the manuscript; David Terrano for maintaining the animal colony; Vytas Bindokas for his help with confocal microscopy; and Christo Goridis, François Guillemot, Kathy Millen, Jeanette Nardelli and Vicky Prince for sending us plasmids for in situ probes. This work was supported in part by the Adler Foundation (A.L. and S.S.S.) and the March of Dimes (E.A.G.).

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