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First published online 26 May 2004
doi: 10.1242/dev.01191

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Presenilin 1 in migration and morphogenesis in the central nervous system

Angeliki Louvi^*,, Sangram S. Sisodia and Elizabeth A. Grove

Department of Neurobiology, Pharmacology and Physiology, University of Chicago, 947 E. 58th Street, Chicago, IL 60637, USA

View larger version (60K): [in a new window]   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 ß-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 Isl1 expression 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.

View larger version (98K): [in a new window]   Fig. 2. Disorganized cerebral cortex in the Psen1 mutant. (A-D) Nissl-stained coronal sections through wild-type (A,C) and Psen1 mutant (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.

View larger version (90K): [in a new window]   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) Cdh6 expression 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 Hes5 expression 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.

View larger version (80K): [in a new window]   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 (100 µm) 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.

View larger version (137K): [in a new window]   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.

View larger version (90K): [in a new window]   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.

View larger version (86K): [in a new window]   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 Rora expression, 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).

View larger version (134K): [in a new window]   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.

View larger version (80K): [in a new window]   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 Gbx2 expression 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.