AbstractWith 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. Developmental Dynamics 236:1273–1286, 2007. © 2007 Wiley‐Liss, Inc.