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A hypomorphic allele of dab1 reveals regional differences in reelin-Dab1 signaling during brain development

Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109, USA

View larger version (38K): [in a new window]   Fig. 1. Expression of Dab1 p80 and p45. (A) Schematic of p80 and p45 protein structures, showing PTB domain, tyrosine phosphorylation sites (p.Tyr), two regions of high sequence identity to Dab2 (DH1 and DH2), and predicted sites for Cdk5 phosphorylation and AP2 binding. (B) SDS PAGE analysis of samples from wild-type, Dab1p45/+ heterozygous and Dab1p45/p45 homozygous cerebral hemispheres sampled at E16.5. Brain lysates were analyzed directly (lanes 3-5) or after immunoprecipitation with anti-Dab1(B3) antibody (lanes 6-11). The 555-codon and 271-codon Dab1 mRNAs were translated in vitro and analyzed in parallel (lanes 1 and 2, respectively). Proteins were detected by western blotting with anti-Dab1(B3) antibody (lanes 1-8) or anti-phosphotyrosine (4G10) (lanes 9-11). Bands below p80 and p45 in lanes 1 and 2 represent internal initiation sites. Bands above p80 and p45 in lanes 3-11 may represent phosphorylated forms. Asterisks mark nonspecific bands. (C) Relative levels of expression of p80 and p45. Dab1p45/WT brains were harvested at P19 and E16.5. Serial threefold dilutions of each brain lysate were analyzed by SDS PAGE and western blotting with anti-Dab1(B3) antibody. (D) Effect of Reln mutation on expression of p80 and p45. Littermate embryos from a Dab1p45/+ Reln–/+ intercross were harvested at E16.5 and cerebral hemispheres were analyzed for Dab1 content. Tubulin was used as a loading control. Genotypes were Dab1+/+ (top two panels) or Dab1p45/p45 (bottom two panels), and wild type, heterozygous or mutant for Reln. Molecular mass standards were 95, 70, 54 and 47 kDa.

View larger version (149K): [in a new window]   Fig. 2. Abnormal development of CA1 region of hippocampus in Dab1p45/– hemizygotes. (A,A’) Dab1p45/–, (B,B’) Dab1p45/+, (C) Dab1p45/p45, (D) Dab15F/5F, (E) Dab1+/– and (F) Dab1p45/p45Reln–/+ brains were sectioned and stained with Nissl. Animals A-C were littermates, and all brains were harvested at P20. The arrow indicates ectopic pyramidal cell layer, squares mark the alveus and circles mark the hippocampal fissure. These provide landmarks for the inner (ventricular) and outer (pial) surface of the hippocampus. Abbreviations: 1, CA1 region; 2, CA2 region; 3, CA3 region; DG, dentate gyrus. Scale bar: 400 µm in A-F; 25 µm in A’,B’.

View larger version (85K): [in a new window]   Fig. 3. Abnormal development of neocortex of Dab1p45/– hemizygotes. (A) Dab1p45/–, (B) Dab1p45/+, (C) Dab1p45/p45, (D) Dab15F/5F, (E) Dab1+/– and (F) Dab1p45/p45Reln–/+ brains were sectioned and stained with Nissl. Arrows indicate ectopic neurons in the marginal zone. Abbreviations: MZ, marginal zone (layer I); II-VI, layers II-VI of the cortical plate. Scale bar: 100 µm.

View larger version (96K): [in a new window]   Fig. 4. Normal preplate splitting in Dab1p45/– hemizygotes. Immunhistochemical detection of chondroitin sulphate proteoglycan (CSPG) in saggital sections of E16.5 brains. (A) Dab1p45/–, (B) Dab1–/+, (C) Dab1p45/p45 and (D) Dab1p45/+ littermate brains. Arrowheads indicate CSPG staining of marginal zone and subplate. Scale bar: 200 µm.

View larger version (65K): [in a new window]   Fig. 5. Late cortical plate neurons in marginal zone of Dab1p45/– hemizygotes. Dab1p45/– and Dab1p45/+ mice were crossed and pregnant females injected with BrdU on E12.5 (A,C) or E16.5 (B,D). (A,B) Dab1p45/–, (C) Dab1+/– and (D) Dab1p45/+ littermates were sacrificed on P20 and BrdU-labeled nuclei detected by immunofluorescence. Note that, in both Dab1p45/– and control, nuclei labeled at E12.5 are deep in layers IV-VI and nuclei labeled at E16.5 are shallow in layers II-III. Neurons labeled at E16.5 migrate into the marginal zone (MZ) in Dab1p45/– but not in controls. Scale bar: 100 µm.

View larger version (43K): [in a new window]   Fig. 6. Reelin response of cultured cerebral neurons. Neurons were cultured from Dab1p45/+ embryos, which express both p80 and p45, and treated with reelin-containing (Reln) or control (C) supernatant from 293T cells. Samples were immunoprecipitated with anti-Dab1(B3) and analyzed by SDS PAGE and western blotting with anti-Dab1(B3) (part A, lanes 6-10) or anti-phosphotyrosine (part A, lanes 1-5 and part B). (A) Incubation with serial fourfold dilutions of reelin-containing supernatant for 15 minutes, or (B) with undiluted reelin-containing supernatant for various times. Asterisks to the right of each panel mark nonspecific bands. Molecular mass standards were 95, 70, 54 and 47 kDa.

View larger version (31K): [in a new window]   Fig. 7. Working models to explain over-migration of late cortical plate neurons and split hippocampus in Dab1p45/–hemizygotes. (A) Positions of different classes of neocortical neurons at birth for wild type (left), Dab1–/– or Reln–/– (center) and Dab1p45/– (right). At this time, the latest cortical plate (CP) neurons (pale yellow) are migrating up radial glia (black) from the ventricular zone (VZ) towards the marginal zone (MZ). Cajal-Retzius neurons (dark green) in the MZ have previously secreted reelin (pale green), although reelin production in the MZ declines after birth. In wild type, reelin excludes CP neurons and stimulates their release from radial glia. Because the early CP neurons (dark yellow) have already been released from the glia, later CP neurons (light yellow) can migrate past them until they reach the MZ. In Dab1–/– or Reln–/–, early CP neurons enter the MZ and only release slowly from radial glia. The ability of late CP neurons to respond to reelin is unknown, because they are held up below the traffic jam caused by the early-born CP neurons. In addition, the subplate neurons (purple) are pushed into the MZ. In Dab1p45/–, early-born CP neurons do release from the radial glia, and the inability of the late CP neurons to respond to reelin is evident from their entry into the MZ. Release of the late CP neurons from radial glia may also be defective, and cause a traffic jam as shown. (B) Migration paths and settling points of pyramidal neurons in the hippocampus. Normally, pyramidal cells (dark blue) settle in the stratum pyramidale (SP), which is proximal to the IMZ in the CA1 region. At the time of arrival of these cells, the IMZ is expressing a low amount of reelin (pale green). In Dab1p45/–, a significant population of pyramidal cells passes through the SP and forms a second layer (open blue symbols). We suggest that this layer may form at the edge of the OMZ, where higher concentrations of reelin are expressed (dark green). As the hippocampus matures, the OMZ becomes the stratum lacunosum moleculaire and the IMZ becomes the stratum radiatum.

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