Molecular Developmental Neurobiology
The research interest of the group is focused on molecular mechanisms for patterning and neurogenesis in the developing and adult brain, with a recent emphasis on the neocortex. The neocortex is a mammal-specific region of the cerebrum, acting as an integrative and executive center, which has a critical importance for the human health. The complex organization of the neocortex arises during development from astonishingly few original cells, the neural stem cells, through precisely controlled phases of cell proliferation, differentiation, migration, and death. How a limited number of multipotent neural stem cells generate the immense cell diversity of the adult brain is one of the challenges in neurobiology.
Using the mouse as a model, we are applying a range of cell and molecular biological, biochemical, neuroanatomical, genetic, and transgenic approaches to investigate how specific genetic programs regulate different aspects of mammalian corticogenesis. Specifically, the role of transcription factors Pax6, Scratch2, and Zbtb20 in the establishment of neuronal subtype identity is currently addressed. In a second direction of research, we are interested in understanding the role of subunit composition of the BAF (SWI/SNF) chromatin remodeling complex in cortical neurogenesis in the embryonic and adult brain. The group also seeks to reveal mechanisms of microRNA-dependent regulation of neurogenesis and brain tumor progression.
Background of our research
Cortical neurogenesis is a complex process in which proliferation and differentiation are coordinated with regionalization, axonal pathfinding, areal and layer specification, as well as with cell death. Recently, we have demonstrated that Ambra1, a gene identified in previously performed gene-trap screen, encodes a novel protein that plays a crucial role in brain development by activating Beclin1-regulated autophagy. Consequently, abrogation of the function of Ambra1 causes an imbalance between cell proliferation and cell death, leading to congenital brain malformations, exencephalus and spina bifida (Fig. 1).
The principal neuronal types of the cortex are the excitatory glutamatergic pyramidal cells, generated by progenitors in the pallial ventricular and subventricular zone. The cortical inhibitory GABA-ergic interneurons are mostly generated in germinal zones of the subpallium, from where they migrate tangentially across the corticostriatal border to reach the cortex. We found that the evolutionarily conserved transcription factor (TF) Pax6 has multiple roles in cortical development, controlling dorsoventral patterning of the embryonic telencephalon, progenitor proliferation, differentiation, establishment of adhesive properties, and axonal projections. For instance, in Pax6 deficiency, a subset of cortical progenitors is molecularly ventralized, generating subcortical interneurons instead of cortical projection neurons causing severe brain malformations (Fig. 2). In the absence of both TFs, Pax6 and Emx2, cortex is not formed and is replaced by tissue with subpallial characteristics.
In the neocortex, neurons born at a specific developmental stage and having distinct morphology, transmitter phenotype, and connectivity, are distributed radially into six layers and tangentially in multiple functional domains, each with its own responsibilities. Neurogenesis in developing cortex progresses from the birth of neurons of the lower layers (L6, L5) at early stages to generation of upper layer (L4 - L2) neurons, produced mostly later in development. Emerging evidence suggests that graded expression of TFs acting in a combinatorial mode generates an areal proto-map in the germinal zones that broadly specifies area identities. Subsequently, expression of downstream molecular determinants in the postmitotic neurons controls the acquisition of final areal identity and shapes the axonal connections. Layer and areal formations are interrelated processes. Consistent with a role of intrinsic mechanisms for cortical regionalization, by combination of microarray and in situ hybridization assays we identified and analyzed the function of a number of genes showing a restricted expression in neuronal subsets of distinct cortical domains and layers, implementing important functions (Fig. 3).
The emerging view suggests that differentiation from stem cells through fated progenitors to differentiated neurons and glial cells proceeds through sequence of stages, associated with timely specific expression of TFs in distinct pathways. We have demonstrated that Pax6 is an intrinsic determinant of the radial glial progenitors, which nowdays are known to produce both neuronal and glial cells. By using Cre-LoxP based approaches for conditional recombination in ES cells and transgenic technology, we have generated mouse lines allowing conditional expression of Pax6 in cortical progenitors. The results from the performed analysis indicated that upon overexpression of Pax6, the outcome (enhanced neurogenesis, misregulation of mitotic cycle or cell death) depends on the endogenous Pax6 dosage in the progenitors of distinct cortical domains (Fig. 4).
Using a similar approach, we found that conditional deletion of Pax6 in cortical progenitors, the adult cortex contains an excess of lower layer (L5) neuronal lineages, generated by the early progenitors, while upper neuronal layers (L4-L2) are almost completely abrogated (Fig. 5). Mechanistically, we found that by regulating the progenitor exit from the mitotic cycle, Pax6 controls the output of the early born lower layer neurons, keeping thereby the progenitor pool for generation of neurons with upper layer identity late in development.
In vivo MRI analysis of the brain of cortex-specific Pax6 knock-out mice (Pax6cKO) revealed disturbances in the cortical organization, indicating a role of Pax6 in neuronal fiber connectivity (Fig. 6). Because of detected selective shrinkage of the motor in expense of the somatosensory and visual areas in the Pax6-deficient Small eye (Sey/Sey) embryos, it was assumed that the cortical areal identity is controlled by the TF Pax6. Thalamocortical tracing in adult conditional Pax6cKO mice revealed, however, that while the graded Pax6 expression in the germinal zone influences the molecular regionalization of the cortex, it does not disturb the establishment of the areal identity (Fig. 7). Furthermore, the generated novel Pax6cKO mouse line showed sensorimotor and cognitive disabilities, similar to deficiencies of aniridia patients with PAX6 defect, thus representing the first mouse model for this human genetic disorder.
Accumulating evidence indicates that the dosage of Pax6 protein in progenitor cells has a pivotal role in corticogenesis and forebrain patterning. Recently, we demonstrated an important role for Trim11 in ubiquitination and proteosome-mediated degradation of Pax6 protein. Our data indicate that an autoregulatory feedback loop between Trim11 and Pax6 exists that assures maintenance of Pax6 protein at a physiological level in cortical progenitors, having an essential role for a normal neurogenesis (Fig. 8).
The cortical neurons are generated through the modes of direct and indirect neurogenesis, the first one producing neurons directly from radial glial cells (RGPs), while the second one through generated intermediate progenitors (IPs) that amplify particular neuronal fates. Our recent studies indicate that conditional activation of TF Scratch2 in cortical progenitors of transgenic mice promotes neuronal differentiation by favoring the direct mode of neurogenesis of RGP, at the expense of IP generation. Moreover, Scratch2 controls neuronal migration by negatively regulating the transcriptional activation of bHLH TFs Ngn2/NeuroD1 on E-box-containing target genes, including Rnd2, recently identified by other groups as a major effector of migrational ability of the neurons (Fig. 9).