Transcriptional regulation of neocortical development

The neocortex is a mammalian-specific brain region that has acquired a unique laminar organization during evolution. The six neocortical layers are each characterized by distinct cellular morphologies, gene expression patterns, axonal connections and functions. This organization contrasts to the cerebral hemispheres of other vertebrate species, which have maintained a simpler, nuclear organization. Despite this morphological divergence, the mammalian neocortex shares several features with the cerebral hemispheres of other vertebrates, including their derivation from progenitor cells in the dorsal telencephalon (pallium), and the expression of a common set of regionalized genes. This conservation suggests that gene orthologs specify similar phenotypes in the embryonic telencephalon in all vertebrates. How embryonic similarities are then translated into the generation of such diverse adult structures is not well understood. To test homology of neocortical layers and pallial nuclei at the level of gene regulatory networks, I performed a comparative analysis of the regulatory elements that govern cerebral gene expression in two evolutionarily distant model organisms: mouse and zebrafish. For this purpose, I validated the technique of in utero electroporation for the functional analysis of regulatory elements in the murine neocortex. I then performed a cross-species comparative analysis of the regulatory sequences that drive layer V-specific Etv1 expression in the murine neocortex, and the nuclear pattern of zebrafish zetv1, identifying conserved elements. This study has led to a better understanding of how genetic and structural modules have co-evolved in the telencephalon (Chapter 3).;Taken together, I have helped to develop a novel technique for efficient analysis of gene regulatory elements and gene function in the developing neocortex. I have used this tool to provide evidence of a functional conservation of cerebral-specific gene regulation between vertebrate species. Moreover, I have demonstrated that Plagl1 plays an important role in regulating mouse neocortical development, in part through its ability to regulate Adcyap1 signalling. While the exact nature of the Plagl1 downstream events remain to be clarified, my studies have laid the groundwork for exciting new discoveries in neocortical development.;The six neuronal layers of the mammalian neocortex are sequentially generated in an inside-out manner during development. Proper laminar positioning of cortical neurons requires coordinated patterns of cell proliferation, neuronal differentiation and migration, processes that are dependent on the synchronized interplay of intrinsic and extrinsic signals. Plagl1 is a maternally imprinted tumour suppressor gene encoding a zinc finger transcription factor that is a key determinant of proliferation and neuronal layering in the developing retina. Plagl1 is also expressed in murine neocortical progenitors and a subset of postmitotic neurons, prompting me to examine its function during mouse corticogenesis (Chapter 4). I found that cortical progenitors misexpressing Plagl1 display significant delays in: 1) exiting the proliferative zones, 2) undergoing progenitor cell maturation, 3) undergoing neuronal maturation and 4) migrating to their appropriate laminar targets. BrdU-pulse labelling, birthdating and marker analyses on Plagl1 mutant neocortices also revealed defects in proliferation and neuronal migration. I also demonstrated that transcript levels of Adcyap1r1, a receptor for the neuropeptide Adcyap1, are regulated by Plagl1 in neocortical progenitors, with altered Adcyap1 signalling accounting in part for the defects in proliferation and cell migration observed in our Plagl1 loss and gain-of-function experiments. Finally, I completed a microarray analysis on Plagl1 -overexpressing cortical cells, identifying several genes that might be regulated by Plagl1 in the developing neocortex, including several transcription factors (e.g. Plag1, Pax6, Neurog1, Insm1, Tbr1, Bcl11b), signalling molecules (e.g. TGFbeta, FGF, Wnt, Trk, Slit-Robo, Semaphorin-Plexin, integrin) and cytoskeletal regulators (e.g. GTPases and modifying enzymes) previously implicated in regulating neocortical cell fate decisions (Chapter 5).