Doublecortin is required in mice for lamination of the hippocampus but not the neocortex

Abstract

Doublecortin (DCX) is a microtubule-associated protein that is required for normal neocortical and hippocampal development in humans. Mutations in the X-linked human DCX gene cause gross neocortical disorganization (lissencephaly or "smooth brain") in hemizygous males, whereas heterozygous females show a mosaic phenotype with a normal cortex as well as a second band of misplaced (heterotopic) neurons beneath the cortex ("double cortex syndrome"). We created a mouse carrying a targeted mutation in the Dcx gene. Hemizygous male Dcx mice show severe postnatal lethality; the few that survive to adulthood are variably fertile. Dcx mutant mice show neocortical lamination that is largely indistinguishable from wild type and show normal patterns of neocortical neurogenesis and neuronal migration. In contrast, the hippocampus of both heterozygous females and hemizygous males shows disrupted lamination that is most severe in the CA3 region. Behavioral tests show defects in context and cued conditioned fear tests, suggesting that deficits in hippocampal learning accompany the abnormal cytoarchitecture.

Fig. 1.

A targeted mutation in theDcx gene eliminates expression of the Dcx protein and results in increased postnatal lethality. A, Targeting strategy. SA-lacZ represents the lacZcoding sequence with an upstream splice acceptor. PGK-neo and PGK-DT are the neomycin and diphtheria toxin genes driven by a phosphoglycerokinase enhancer. Animals were typed by Southern blotting using a PCR product (probe A). The correct configuration of the targeted locus was also confirmed by Southern blotting using a second DNA fragment (probe B; data not shown). E, EcoRI;N, NheI; EV,EcoRV (only the relevant restriction endonuclease sites are indicated). B, Southern blot analysis of knock-out animals. Probe A was hybridized to genomic DNA from Dcxhemizygous mutant (−/Y), wild-type (wt), and heterozygous (−/+) animals (offspring of a heterozygous female and a wild-type male) that was digested with EcoRI. The 6.5 and 8.3 kb bands represent the wild-type and mutant alleles, respectively (see A).C, Western blot analysis of P0 mutant brains. Note the total absence of reactivity with the anti-Dcx antibody (11) in the hemizygous brain and approximately half normal quantity in the heterozygous brain. The blot was stripped and reprobed with an antibody against glutamate decarboxylase (α-GAD) as a loading control. D, Progeny of crosses between a Dcx −/+ female and a wild-type male. Note that the hemizygous males are present in an approximately Mendelian ratio (2:1:1) at P0 but that their relative numbers are reduced by adulthood. The P0 and adult figures represent two separate cohorts. E, Immunostaining of E15 brains with anti-Dcx antibody (top) and histochemical reaction of 5-week-old brains with X-gal (bottom). Note the total absence of Dcx reactivity in the mutant brain. The focal red signal in the mutant represents nonspecific background staining of blood vessels and leptomeninges. X-gal staining demonstrates perdurant β-galactosidase activity predominantly in layers 2–4 in the mutant brain but none in the wild type.

Fig. 2.

Dcx mutant mice show normal brain morphology. A, The adult Dcx mutant brain is strikingly normal, with the exception of abnormal layering in the hippocampus. B, The Dcx mutant neocortex shows the normal six-layered pattern. Note the similarity in thickness of the laminas and the density of cell populations between mutant and wild type (wt). C, Dcxmutant cerebellum is indistinguishable from wild type. Note the normal overall morphology of the cerebellum (insets) as well as the normal layering pattern of the cerebellar cortex: molecular layer, Purkinje cell layer, and internal granular layer. D,Dcx mutant retina shows normal layering.

Fig. 3.

Analysis of cortical layering in theDcx mutant mouse using the thy1–YFP marker. Top, The brains of adult wild-type (wt) and Dcx mutant (−/Y) mice into which has been crossed a thy1–YFP transgene (YFP-H in Feng et al., 2000) that is expressed predominantly in a subset of layer 5 neurons as well as in pyramidal cells of the hippocampus and cells of the fascia dentata. Close-up images of the mutant neocortex (bottom) show a distribution of YFP-expressing cells in layer 5 similar to that in the wild type. Note that scattered YFP-expressing cells are apparent in layer 3 (arrowhead) in the wild type. Such cells were identified with similar frequency in the mutant as well but are not present in the particular field pictured.

Fig. 4.

Embryonic development of the neocortex inDcx mutant mice. A, Cresyl violet-stained coronal sections of E15 brains (top). Note that theDcx mutant (−/Y) brain is indistinguishable from wild type (wt). Coronal sections of E15 brains immunostained with an antibody against TuJ1 are shown at the bottom. This antibody labels postmitotic migrating and differentiating neurons and is coexpressed with Dcx (Francis et al., 1999). Note the very similar pattern of staining between mutant and wild type. B, BrdU birth dating of cortical neurons in Dcx mutants. Brains were harvested at P0 from mice born to dams that had received intraperitoneal injections of BrdU either on E12 (E12→P0) or E14 (E14→P0) and were stained for BrdU. Note that in the brains from animals injected at E12 the labeled cells are more concentrated in the lower layers but are present throughout the cortex. In contrast, in brains from animals injected at E14 there is a tighter clustering of labeled cells in the upper layers of the cortex. The distribution of cells does not differ significantly between wild type and mutant.

Fig. 5.

Abnormal hippocampal lamination in theDcx mutant. A, Wild-type (wt) and Dcx mutant hippocampi stained with the neuronal marker NeuN. Note the greater degree of disorganization in the pyramidal layer of the Dcx mutant (−/Y) compared with the wild type. Although the disorder is greatest in area CA3 (bottom), a looser layering of cells is also readily appreciable in area CA1 (middle). B, Area CA3 in wild type,Dcx heterozygotes (−/+), andDcx hemizygotes (−/Y). Note the partial splitting of the pyramidal cell layer (top,black arrowhead) and disorganized lamination (white arrowhead) in the hemizygous brain. The heterozygote demonstrates milder but similar defects. Analysis of thethy1–YFP transgene in the heterozygote and homozygote shows a progressively greater disorganization of the YFP-expressing cells relative to wild type in area CA3 of the hippocampus (bottom). C, D, Confocal images of hippocampal area CA3. C, In the areas of CA3 in which lamination is relatively preserved in the mutant, dendritic processes appear to be indistinguishable from wild type. D, This is also true in areas that show a greater degree of disorganization in the mutant. The wider spacing between labeled neurons in theDcx mutant is secondary to greater scattering of the cell bodies.

Fig. 6.

Dcx mutant mice show defects in strength and hippocampal-based learning.A, Cage-top hang test in a mixed (129/SvJ × NIH Black Swiss) background (t₍₁₈₎ = 2.75;p < 0.01). B, Wire hang test in a 129/SvJ background (t₍₁₀₎ = 9.7;p < 0.001). C, Cued conditioned fear test in a mixed background (t₍₁₆₎= 4.94; p < 0.0001). D, Context conditioned fear test in a 129/SvJ background (t₍₁₀₎ = 3.5; p < 0.01).