In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

Abstract

SummaryBrain structures such as the outer subventricular zone (OSVZ) and the inner fiber layer (IFL) in the developing cerebral cortex are especially prominent in higher mammals. However, the molecular mechanisms underlying the formation of the OSVZ are still largely unknown, mainly because genetic manipulations that can be applied to the OSVZ in higher mammals had been poorly available. Here we developed and validated a rapid and efficient genetic manipulation technique for germinal zones including the OSVZ using in utero electroporation in developing gyrencephalic carnivore ferrets. We also determined the optimal conditions for using in utero electroporation to express transgenes in germinal zones. Using our electroporation procedure, the morphology of GFP-positive cells in the OSVZ was clearly visible even without immunostaining, and multiple genes were efficiently co-expressed in the same cells. Furthermore, we uncovered that fibers, which seemed to correspond to those in the IFL of monkeys, also existed in ferrets, and were derived from newly generated cortical neurons. Our technique promises to be a powerful tool for investigating the fundamental mechanisms underlying the formation and abnormalities of the cerebral cortex in higher mammals.

Human-specific genomic signatures of neocortical expansion

“…These insights are complemented, on the one hand, by advances in novel transgenic, primate and non-primate, animal models, such as the gyrencephalic ferret [95], the marmoset [96] or the macaque [97]. On the other hand, 3D cerebral organoids generated from human and non-human primate iPSCs provide promising tools to dissect evolutionary differences in cortical development in vitro.…”

YAP activity is necessary and sufficient for basal progenitor abundance and proliferation in the developing neocortex

“…We performed IUE of ferret embryos to deliver the DN-YAP construct into the developing ferret neocortex (Kawasaki et al, 2012;Kawasaki et al, 2013). Specifically, we coelectroporated the ferret dorsolateral neocortex with either CAGGS-empty vector plus CAGGS-EGFP or with CAGGS-DN-YAP plus CAGGS-EGFP.…”

“…In utero electroporation of E33 ferret embryos was performed as originally established (Kawasaki et al, 2012;Kawasaki et al, 2013), with the modifications indicated below.…”

Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue

“…Electroporation allows for genetic manipulation of neural stem and progenitors cells in a relatively short time window and is therefore less time consuming than transgenesis [4][5][6] . In addition, electroporation of the developing neural tube is very versatile: it can be adapted to different species (mouse, rat and ferret [7][8][9] ) and allows for different types of manipulation, ranging from overexpression of a specific gene to gene knockdown 5 . As electroporation relies on the application of an electric field, only charged molecules can be delivered, thus limiting the spectrum of molecules that can be used.…”