Malformations of cortical development are characteristic of a plethora of diseases that includes polymicrogyria, periventricular and subcortical heterotopia and lissencephaly. Mutations in TUBA1A and TUBB2B, each a member of the multigene families that encode α- and β-tubulins, have recently been implicated in these diseases. Here we examine the defects that result from nine disease-causing mutations (I188L, I238V, P263T, L286F, V303G, L397P, R402C, 402H, S419L) in TUBA1A. We show that the expression of all the mutant proteins in vitro results in the generation of tubulin heterodimers in varying yield and that these can co-polymerize with microtubules in vitro. We identify several kinds of defects that result from these mutations. Among these are various defects in the chaperone-dependent pathway leading to de novo tubulin heterodimer formation. These include a defective interaction with the chaperone prefoldin, a reduced efficiency in the generation of productive folding intermediates as a result of inefficient interaction with the cytosolic chaperonin, CCT, and, in several cases, a failure to stably interact with TBCB, one of five tubulin-specific chaperones that act downstream of CCT in the tubulin heterodimer assembly pathway. Other defects include structural instability in vitro, diminished stability in vivo, a compromised ability to co-assemble with microtubules in vivo and a suppression of microtubule growth rate in the neurites (but not the soma) of cultured neurons. Our data are consistent with the notion that some mutations in TUBA1A result in tubulin deficit, whereas others reflect compromised interactions with one or more MAPs that are essential to proper neuronal migration.

The high-resolution structure of doublecortin-stabilized microtubules provides unprecedented insight into their in vivo architecture.

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs.

Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence, were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to further study how lamination defects underlie the genesis of epileptiform activities.

This review intends to provide examples how comparative and genetic analyses both contribute to our understanding of the rules for cortical development and evolution. Genetic studies helped to understand evolutionary rules of telencephalic organization in vertebrates. The control of the establishment of conserved telencephalic subdivisions and the formation of boundaries between these subdivisions has been examined and revealed the very specific alterations at the striatocortical junction. Comparative studies and genetic analyses both demonstrated the differential origin and migratory pattern of the two basic neuron types of the cerebral cortex. GABAergic interneurons are mostly generated in the subpallium and a common mechanisms govern their migration to the dorsal cortex in both mammals and sauropsids. The pyramidal neurons are generated within the cortical germinal zone and migrate radially. The earliest generated cell layers comprising preplate cells. Reelin positive Cajal-Retzius cells are a general feature of all vertebrates studied so far, however, there is a considerable amplification of the reelin signaling, which might have contributed to the establishment of the basic mammalian pattern of cortical development. Based on numerous recent observations we shall present an argument that specialization of the mitotic compartments might constitute a major drive behind the evolution of the mammalian cortex. Comparative developmental studies revealed distinct features in the early compartments of the developing macaque brain drawing our attention to the limitations of some of the current model systems for understanding human developmental abnormalities of the cortex. Comparative and genetic aspects of cortical development both reveal the workings of evolution.

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neoR- and hprt-expression cassettes from three different tri-lox mutants.