Functional redundancies, compensatory mechanisms, and lethal phenotypes often prevent the full analysis of gene functions through generation of germline null mutations in the mouse. The use of site-specific recombinases, such as Cre, which catalyzes recombination between loxP sites, has allowed the engineering of mice harboring targeted somatic mutations, which are both temporally controlled and cell-type restricted. Many Cre-expressing mouse lines exist, but only a few transgenic lines are available that harbor a reporter gene whose expression is dependent on a Cre-mediated event. Moreover, their use to monitor gene ablation at the level of individual cells is often limited, as in some tissues the reporter gene may be silenced, be affected by position-effect variegation, or reside in a chromatin configuration inaccessible for recombination. Thus, one cannot validly extrapolate from the expression of a reporter transgene to an identical ablation pattern for the conditional allele of a given gene. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending on the orientation of the flanking loxP sites, and the availability of both wild-type (WT) and mutant loxP sites, we designed a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on. We demonstrate the efficiency and reliability of this switch to readily detect, in the mouse, at the single cell level, Cre-mediated gene ablation. We discuss how this strategy can be used to generate genetic modifications in a conditional manner.
To explore the function of genes expressed by myelinating cells we have developed a model system that allows for the inducible ablation of predetermined genes in oligodendrocytes and Schwann cells. The Cre/loxP recombination system provides the opportunity to generate tissue-specific somatic mutations in mice. We have used a fusion protein between the Cre recombinase and a mutated ligand-binding domain of the human estrogen receptor (CreER(T)) to obtain inducible, site-specific recombination. CreER(T) expression was placed under the transcriptional control of the regulatory sequences of the myelin proteolipid protein (PLP) gene, which is abundantly expressed in oligodendrocytes and to a lesser extent in Schwann cells. The CreER(T) fusion protein translocated to the nucleus and mediated the recombination of a LacZ reporter transgene in myelinating cells of PLP/CreER(T) mice injected with the synthetic steroid tamoxifen. In untreated animals CreER(T) remained cytoplasmic, and there was no evidence of recombination. The PLP/ CreER(T) animals should be very useful in elucidating and distinguishing a particular gene's function in the formation and maintenance of the myelin sheath and in analyzing mature oligodendrocyte function in pathological conditions.
The link between cortical precursors G1 duration (TG1) and their mode of division remains a major unresolved issue of potential importance for regulating corticogenesis. Here, we induced a 25% reduction in TG1 in mouse cortical precursors via forced expression of cyclin D1 and cyclin E1. We found that in utero electroporationmediated gene transfer transfects a cohort of synchronously cycling precursors, necessitating alternative methods of measuring cell-cycle phases to those classical used. TG1 reduction promotes cell-cycle reentry at the expense of differentiation and increases the self-renewal capacities of Pax6 precursors as well as of Tbr2 basal precursors (BPs). A population level analysis reveals sequential and lineage-specific effects, showing that TG1 reduction: (i) promotes Pax6 self-renewing proliferative divisions before promoting divisions wherein Pax6 precursors generate Tbr2 BPs and (ii) promotes self-renewing proliferative divisions of Tbr2 precursors at the expense of neurogenesis, thus leading to an amplification of the BPs pool in the subventricular zone and the dispersed mitotic compartment of the intermediate zone. These results point to the G1 mode of division relationship as an essential control mechanism of corticogenesis. This is further supported by longterm studies showing that TG1 reduction results in cytoarchitectural modifications including supernumerary supragranular neuron production. Modeling confirms that the TG1-induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. These findings also have implications for understanding the mechanisms that have contributed to brain enlargement and complexity during evolution.basal progenitor ͉ cell-cycle ͉ corticogenesis C ortical areas are characterized by their cytoarchitecture, an expression of the morphology and density of their constituent neurons. Areal differences in neuron number and phenotype are distinguishing features both within and across species (1, 2). The developmental processes that specify the number of neurons and their laminar fate are therefore instrumental in specifying cortical cytoarchitecture. Neuron number in layers and areas correlate with changes in the rate of neuron production, largely determined by the balance between cell-cycle reentry and exit (3, 4). Proliferative division generates two progenitors that re-enter the cell-cycle, whereas differentiative division gives rise to at least one daughter cell that undergoes differentiation. An open question is how the decision between proliferative versus differentiative division is made (5).Key observations suggest a concerted regulation of TG1 and mode of division. During mouse corticogenesis, a progressive increase in rates of neuron production, is accompanied by increasing frequencies of differentiative divisions, and a slowing down of TG1 (6). Proliferative divisions are characterized by short TG1 and differentiative divisions by long TG1 (3, 7-9). G1 represents a critical phase during cell-cycle progression, wh...
Friedreich ataxia and ataxia with selective vitamin E deficiency (AVED) share very similar clinical phenotypes. We have mapped the AVED locus to proximal 8q with only three large consanguinous Tunisian families, representing to our knowledge the first use of homozygosity mapping for primary linkage analysis. Subsequently, three additional families showed linkage with the same markers. A maximum lod score of 17.9 was obtained at theta = 0 for the haplotype D8S260-D8S510, consisting of the two closest markers. With only 6 families, the AVED locus is therefore mapped precisely as illustrated by the lod-1 confidence interval of 2.4 cM on either side of D8S260-D8S510. Isolation of a yeast artificial chromosome contig > 800 kilobases (kb) showed that D8S260 and D8S510 are less than 400 kb apart.
Summary Major non primate-primate differences in corticogenesis include the dimensions, precursor lineages and developmental timing of the germinal zones (GZ). microRNAs (miRNAs) of laser dissected GZ compartments and cortical plate (CP) from embryonic E80 macaque visual cortex were deep sequenced. The CP and the GZ including Ventricular Zone (VZ), outer and inner subcompartments of the Outer SubVentricular Zone (OSVZ) in area 17 displayed unique miRNA profiles. miRNAs present in primate, but absent in rodent, contributed disproportionately to the differential expression between GZ sub-regions. Prominent among the validated targets of these miRNAs were cell-cycle and neurogenesis regulators. Co-evolution between the emergent miRNAs and their targets suggested that novel miRNAs became integrated into ancient gene circuitry to exert additional control over proliferation. We conclude that multiple cell-cycle regulatory events contribute to the emergence of primate-specific cortical features, including the OSVZ, generated enlarged supragranular layers, largely responsible for the increased primate cortex computational abilities.
Christophe, Elodie, Nathalie Doerflinger, Daniel J. Lavery, Zoltán Molnár, Serge Charpak, and Etienne Audinat. Two populations of layer V pyramidal cells of the mouse neocortex: development and sensitivity to anesthetics. J Neurophysiol 94: 3357-3367, 2005. First published July 6, 2005 doi:10.1152/jn.00076.2005. Previous studies have shown that layer V pyramidal neurons projecting either to subcortical structures or the contralateral cortex undergo different morphological and electrophysiological patterns of development during the first three postnatal weeks. To isolate the determinants of this differential maturation, we analyzed the gene expression and intrinsic membrane properties of layer V pyramidal neurons projecting either to the superior colliculus (SC cells) or the contralateral cortex (CC cells) by combining whole cell recordings and single-cell RT-PCR in acute slices prepared from postnatal day (P) 5-7 or P21-30 old mice. Among the 24 genes tested, the calcium channel subunits ␣1B and ␣1C, the protease Nexin 1, and the calcium-binding protein calbindin were differentially expressed in adult SC and CC cells and the potassium channel subunit Kv4.3 was expressed preferentially in CC cells at both stages of development. Intrinsic membrane properties, including input resistance, amplitude of the hyperpolarization-activated current, and action potential threshold, differed quantitatively between the two populations as early as from the first postnatal week and persisted throughout adulthood. However, the two cell types had similar regular action potential firing behaviors at all developmental stages. Surprisingly, when we increased the duration of anesthesia with ketamine-xylazine or pentobarbital before decapitation, a proportion of mature SC cells, but not CC cells, fired bursts of action potentials. Together these results indicate that the two populations of layer V pyramidal neurons already start to differ during the first postnatal week and exhibit different firing capabilities after anesthesia.
Adrenoleukodystrophy (ALD) is a demyelinating disease of the central nervous system that results from a genetic deficiency of ALDP, an ABC protein involved in the transport of very long-chain fatty acids (VLCFAs). The cloning of the ALD gene and the positive effects of allogeneic bone marrow transplantation support the feasibility of a gene therapy approach. We report the retroviral transfer of the ALD cDNA to peripheral blood and bone marrow CD34+ cells from control donors and ALD patients. Prestimulation of these cells with cytokines, followed by infection with the M48-ALD retroviral vector, resulted in 20% transduction efficiency (4-40%) and expression of the vector-encoded ALDP in 20% of CD34+ cells (7.3-50%). Long-term culture (LTC) of transduced CD34+ cells from two ALD patients showed efficient transduction (24-28%) and stable expression (25-32%) of ALDP in derived clonogenic progenitors at 3 weeks of culture. The expression of ALDP in CFU cells derived from 5 and 6 weeks of LTC confirmed the effective transduction of LTC-initiating cells. Expression of ALDP was observed in CD68+ CFU-derived cells, suggesting that monocyte-macrophages, the target bone marrow cells in ALD, were produced from transduced progenitor cells. VL-CFA content was corrected in LTC and CFU-derived cells in proportion to the percentage of transduced cells, indicating that the vector-encoded ALDP was functional. Although not efficient yet to allow a clinical perspective, these results demonstrate the feasibility of ALD gene transfer into CD34+ cells of ALD patients.
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