Validating In Utero Electroporation for the Rapid Analysis of Gene Regulatory Elements in the Murine Telencephalon

Sapienza University of Rome, Roma, Latium, Italy
Developmental Dynamics (Impact Factor: 2.38). 05/2007; 236(5):1273-86. DOI: 10.1002/dvdy.21126
Source: PubMed


With the ultimate goal of understanding how genetic modules have evolved in the telencephalon, we set out to modernize the functional analysis of cross-species cis-regulatory elements in mouse. In utero electroporation is rapidly replacing transgenesis as the method of choice for gain- and loss-of-function studies in the murine telencephalon, but the application of this technique to the analysis of transcriptional regulation has yet to be fully explored and exploited. To empirically define the developmental stages required to target specific populations of neurons in the dorsal telencephalon, or pallium, which gives rise to the neocortex in mouse, we performed a temporal and spatial analysis of the migratory properties of electroporated versus birth-dated cells. Next, we compared the activities of two known Ngn2 enhancers via transgenesis and in utero electroporation, demonstrating that the latter technique more faithfully reports the endogenous telencephalic expression pattern observed in an Ngn2lacZ knock-in line. Finally, we used this approach to test the telencephalic activities of a series of deletion constructs comprised of the zebrafish ER81 upstream regulatory region, allowing us to identify a previously uncharacterized enhancer that displays cross-species activity in the murine piriform cortex and lateral neocortex, yet not in more medial domains of the forebrain. Taken together, our data supports the contention that in utero technology can be exploited to rapidly examine the architecture and evolution of pallial-specific cis-regulatory elements.

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Available from: Carol Schuurmans, Oct 13, 2015
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    • "Because the netrin-1/DCC signaling axis is important for corpus callosum formation (Izzi and Charron, 2011; Fothergill et al., 2014), we next examined the role of Hsc70 on netrin-1–de- pendent axonal projections in vivo. Embryonic telencephalons were transfected with pCIG2 vectors expressing GFP alone or GFP along with Hsc70 or Hsc70 D10N by in utero electroporation at E14.5, when cortical progenitors exclusively generate upper layer commissural projection neurons (Langevin et al., 2007). After electroporation, embryos were left to develop for 3 d in utero before collection and processing. "
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    ABSTRACT: During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1–induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase–deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1–induced Rac1 activation. Hsc70 was required for netrin-1–mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance.
    The Journal of Cell Biology 08/2015; 210(5):817-832. DOI:10.1083/jcb.201505084 · 9.83 Impact Factor
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    • "brain sections (96 ± 36 and 18 ± 7). Considering that expression of detectable levels of EGFP is restricted to cells born shortly after in utero electroporation was performed (36) these observations suggest that the Thy1.2→hGFAP Co-Driver pair targeted reporter gene activation mainly to cells that were born later relative to the tdTomato+ cells observed as a result of hGFAP→Thy1.2 "
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    ABSTRACT: Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.
    Nucleic Acids Research 01/2014; 42(6). DOI:10.1093/nar/gkt1361 · 9.11 Impact Factor
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    • "Distinct neurodevelopmental events in a subset of neurons within a particular region can now be controlled using cell-type-specific regulatory elements (Matsuda and Cepko, 2007; LoTurco et al., 2009; Manent et al., 2009). Cell-type-specific promoter or regulatory elements such as Thy1, Nestin, Tα1, BLBP, PDGF, GLAST (Gal et al., 2006), CAMKII, Synapsin, DCX (Wang et al., 2007b), NSE but also ER81 and Ngn2 (Langevin et al., 2007) can drive expression of a fluorescently labeled construct into a particular neuronal cell population in which the time of expression can be both dependent on the time of electroporation and the period of expression of the regulatory element (Figure 3A). In this way endophenotypes can be further unraveled because of the specificity of the gene manipulation. "
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    ABSTRACT: We have only just begun to decipher the complexity of our brain, including its maturation. Correct brain development and communication among brain areas are crucial for proper cognitive behavior. Brain area-specific genes expressed within a particular time window direct neurodevelopmental events such as proliferation, migration, axon guidance, dendritic arborization, and synaptogenesis. These genes can pose as susceptibility factors in neurodevelopmental disorders eventually resulting in area-specific cognitive deficits. Therefore, in utero electroporation (IUE)-mediated gene transfer can aid in creating valuable animal models in which the regionality and time of expression can be restricted for the targeted gene(s). Moreover, through the use of cell-type-specific molecular constructs, expression can be altered in a particular neuronal subset within a distinct area such that we are now able to causally link the function of that gene in that brain region to the etiology of the disorder. Thus, IUE-mediated gene transfer is an attractive molecular technique to spatiotemporally address the developmental aspects of gene function in relation to neurodevelopmental disorder-associated endophenotypes.
    Frontiers in Molecular Neuroscience 11/2011; 4:37. DOI:10.3389/fnmol.2011.00037 · 4.08 Impact Factor
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