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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02209

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Massive loss of Cajal-Retzius cells does not disrupt neocortical layer order

¹ Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637, USA.
² Division of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University 2-2-1 Honjo, Kumamoto, Kumamoto 860-0811, Japan.
³ Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.

View larger version (73K): [in a new window]   Fig. 1. Constructing Wnt3a-IRES-Cre mouse lines to fate map the hem. (A, top to bottom) Wild-type allele of Wnt3a; the targeting vector containing an IRES-Cre cassette; the targeted Wnt3a-IRES-Cre allele with Cre inserted into the 3' end of the Wnt3a locus. (B,C) Coronal sections through E12.5 forebrains show Cre expression in the hem of Wnt3a-IRES-Cre mice (C), indistinguishable from expression of endogenous Wnt3a (B).

View larger version (70K): [in a new window]   Fig. 2. Cells derived from the hem fit the criteria of Cajal-Retzius cells. (A-C) From E10.5-12.5, hem-derived ß-gal-labeled cells (arrows) migrate into the MZ. (D,E) E18.5 cortical hemispheres in rostral (D) and lateral (E) views. ß-Gal-expressing cells cover the neocortex with rough boundaries at the olfactory bulbs and future rhinal sulcus (arrows). Caudally, some cells migrate below the latter boundary (asterisk). (F,G) p73 immunofluorescence in cells of the hem, the cpx and the MZ of the hippocampal (F) and neocortical primordia (G). Asterisks in F,G indicate regions also shown at higher magnification (insets). Many cells are double-labeled with ß-gal, indicating their origin in the hem (arrows, and higher magnification insets). A few were positive only for p73 (for example, see arrowhead in G). (H,I) Hem-derived, ß-gal+ cells in the neocortex (H) and hippocampus (I) were reelin-immunoreactive (arrows), although some reelin-positive cells were single-labeled (arrowhead in I). (J,K) Hem-derived cells were not immunoreactive for GABA. cpx, choroid plexus; ncx, neocortex; ob, olfactory bulb; reln, reelin.

View larger version (58K): [in a new window]   Fig. 3. Constructing a Wnt3a-IRES-xneox-dt-a mouse line to ablate the hem. (A, top to bottom) Wild-type Wnt3a allele; Wnt3a-IRES-xneox-dt-a allele; activation of dt-a toxin by Cre-recombination, using an Emx1-IRES-Cre mouse line (Gorski et al., 2002). (B) Recombination in the telencephalon was confined to the hem, which was almost completely ablated by E10 in Wnt3aIRESxneoxdt-a/+;Emx1IRESCre/+ mice (dorsal views). Wnt3a expression marks the hem at E12.5 (medial view) but not in a hem-ablated brain (arrow indicates caudal hem remnant). Expression of Lmx1a marks the hem and cpx; a part of the cpx (arrow) remains in the hem-ablated mouse.

View larger version (85K): [in a new window]   Fig. 4. Ablation of the hem leads to loss of CR cells. (A-H) E12.5 forebrains processed with in situ hybridization to show expression of reelin (Reln) or p73 in control (A,C,E,G) or hem-ablated (B,D,F,H) mice. Hemispheres are viewed from the lateral (A,B,E,F) or medial face (C,D,G,H). p73- and Reln-expressing cells cover the cerebral cortex of controls, but are almost absent from medial cortex and lateral neocortex in hem-ablated animals. Ventrolateral (vl) and retrobulbar (rb) expression of Reln is more striking in the absence of neocortical Reln. Cells appear to stray from the rb into dorsomedial cortex (arrows, F,H). A lateral line of p73 expression in hem-ablated cortex (arrow, B) roughly marks the boundary of major CR cell migration from the hem.

View larger version (94K): [in a new window]   Fig. 5. Other sources provide only transient or sparse Reln+ cells to the hem-ablated neocortex. (A-E) E13.5 cerebral hemispheres from control (E) or hem-ablated (A-D) mice, processed with in situ hybridization to show Reln or p73 expression. (A,D,E) Lateral views of hemispheres, rostral to the left; (B,C) dorsal views, rostral is downwards. (A-E) In hem-ablated cortex, ventral (A) and rostral (B) sources appear to provide Reln+ cells (arrows in A,B) to the neocortex. Dorsal cells that may originate from the rostral source do not express p73 (arrow in C), and thus do not fit current criteria for CR cells. In a control (E), Reln+ CR cells cover lateral neocortex; in a hem-ablated mouse, the ventral source provides few Reln+ cells (arrows in D). (F-K) Coronal sections through E12.5, E13.5 and E15.5 hemispheres, medial towards the right, processed to show Reln expression (arrows). Evidence for a rostromedial source of Reln+ neurons (arrows) in sections at E12.5 in both control (F) and hem-ablated brains (I). In the latter, a small domain of Reln+ cells remains in medial cortex at E13.5 (arrow, J), but not at E15.5 (K).

View larger version (117K): [in a new window]   Fig. 6. Diffuse expression of Reln in control and hem-ablated mice. (A-C) Sagittal sections through E15.5 brains; (D-F) coronal sections through E18.5 neocortex. At E15.5 and E18.5 in control mice (A,D), but not hem-ablated mice (B,E), a dense layer of Reln+ CR cells fills the MZ (asterisk in B indicates the region of hem loss and consequent failure of hippocampal development). Below this is more diffuse Reln expression. At E15.5, a band of low Reln+ expression appears in the IZ/SVZ in both mutants and controls (A-C). By E18.5, scattered Reln+ cells appear in layers IV/V (D-F). (F) Layers defined by expression of Rorb: Reln+ cells are yellow, Rorb expression is light purple. Arrows in each panel indicate Reln+ cells.

View larger version (135K): [in a new window]   Fig. 7. In the absence of CR cells the preplate splits and neocortical layers are ordered correctly. (A-F) Coronal sections through control or hem-ablated neocortex. At E13.5, calretinin and CSPG-immunoreactivity (IR) distinguished the preplate (A,C). By E15.5, in control mice, two bands of calretinin- and CSPG-IR marked the MZ and subplate (B,D). Only the lower, subplate band appeared in hem-ablated animals; the upper band, which labeled CR cells in controls, was not detected (B,D). (E,F) In control neocortex at E18.5, expression patterns of SCIP (Pou3f1) SorLA (Sorl1), Rorb, ER81 (Etv1), Fezl and Tbr1 distinguished layers II/III, IV, V, VI and the subplate, along the pial-to-ventricular axis (E). In hem-ablated mice, expression of each gene appeared in a highly similar pial to ventricular order (F). Expression of some genes, notably SorLA, showed abnormal diffusion, indicating modest migration defects.