Validating in utero electroporation for the rapid analysis of gene regulatory elements in the murine telencephalon

Abstract

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.

Convergent repression of Foxp2 3′UTR by miR-9 and miR-132 in embryonic mouse neocortex: implications for radial migration of neurons

“…We found comparable distributions of targeted cells in control cortices and in cortices electroporated with pCAGGS-Foxp2-3ЈUTR-WT (Fig. 5A,G,I), consistent with previous studies (De Pietri Tonelli et al, 2006;Langevin et al, 2007). By contrast, in cortices electroporated with pCAGGS-Foxp2-⌬-3ЈUTR and pCAGGS-Foxp2-3ЈUTR-MT1+2+3, we found that many of the targeted cells were misplaced in the IZ (Fig.…”

“…3A). Consistent with previous reports (De Pietri Tonelli et al, 2006;Langevin et al, 2007), analysis of fluorescence in electroporated brains 8, 48 or 4 days after electroporation confirmed that expression of mCherry was restricted to the population of targeted neural progenitors in VZ/SVZ (Fig. 3B), or was located in both targeted progenitors and neurons that were generated by them (Fig.…”

Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1

“…In utero electroporation was performed on pregnant CD-1 mice at E13 according to published protocols (Langevin et al, 2007;Saito and Nakatsuji, 2001). Embryos were collected at 20 hours, 48 hours and 5 days following in utero electroporation, or 2 weeks postnatal.…”

“…a significant percentage of mt Mcl1-transfected NPCs were already in the CP and (2) GFP and transgene expression from the pCIG2 plasmid are observed by 9 h.p.e. (Langevin et al, 2007). Pregnant dams received an intraperitoneal injection of BrdU at 12 h.p.e.…”

“…E14 therefore marks the transition from generating layer V to layer IV neurons (Takahashi et al, 1999). Since we performed in utero electroporation at E13 and NPCs begin expressing GFP within 9 hours (Langevin et al, 2007), the first cohort of NPCs to be born would be late on E13 or in the very early hours of E14 and, therefore, these cells can contribute to the deeper layers.…”