Fig.1 drawing of the human embryo at 7 gestational weeks.
Fig.1 Drawing of the human embryo at 7 gestational weeks. Boxed area presented on the right with nuclear stain-arrows show mitotic figures on the ventricular surface.

The major focus of my research is cellular development of the cerebral cortex. The two main cell types found in the central nervous system, glia  and neurons, are interconnected and we are equally interested in both cell types. We are particularly focused on molecular characteristics and cell fate determinants of progenitor cells, as this is important to create potential future therapies using these cells.

Fig. 2 Human radial glia cells
Fig. 2 Human radial glia cells enriched by antibody to surface marker Lex (CD15)- green, co-labeled with radial glia marker BLBP (red).

A large repertoire of cortical progenitor cells  may in part explain the unique complexity of the human cerebral cortex. Radial glia cells (RGC) in the human cortical proliferative zones are multipotent progenitors that can generate all major neural cells (neurons, astrocytes and oligodendrocytes). We established organotypic slice cultures and dissociated cell cultures of the human fetal brains, which made experimental studies feasible. Using this approach, we would like to answer following questions: a) What determines the fate of cortical progenitor cells? b) What is the role of transcription factors such as Pax6, Nkx2.1and Olig1,2 in these processes? To study RGCs and their progeny, we transfect cells from cortical proliferative zone with plasmids or viruses that can specifically label RG cells. Progeny of labeled cell is identified with specific neuronal markers and studied electrophysiologically (in collaboration with Dr. Antic from our Department).

We study progenitors of cortical interneurons (GABAergic cells) by single and double-immunolabeling with cell-type specific markers combine with markers of cell proliferation (Ki67, BrdU). Recently we demonstrated  the distribution of ventral transcription factor Nkx2.1, in the human fetal cerebral cortex at mid-gestation (20gw). This unexpected localization  suggests local origin of a subtype of cortical interneurons. We determined that, unlike in rodents, cortical interneurons in the human fetal brain have dual origin. The significance of this finding is that these human-specific cortical interneurons that are generated in the cortex cannot be studied in animal models, and they might be involved in diseases such as schizophrenia or autism (Fig.3, Radonjic et al., Cell Reports 2014c; Selemon and N.Zecevic 2015). 

Figure 3. The expression of Nkx2.1 transcription factor in the human forebrain
Figure 3. The expression of Nkx2.1 transcription factor in the human forebrain. (A-C), Drawing of a coronal section of 20 GW forebrain with distribution pattern of immunolabeled Nkx2.1+ cells. (B) MGE and cortical VZ/SVZ. (C) In situ (FISH) signal for mRNA Nkx2.1 (red) in the whole cerebral cortex at mid-term. (D) In situ hybridization (red) and immunolabeling with Nkx2.1 antibody (green) show co-localization of mRNA and protein. (E) mRNA Nkx2.1 (red) and Gad67 immunolabeling (green) in the same cells; (F) mRNA Gad67 (red) and Nkx2.1 immunolabeling (green) in the same cells. Graphs demonstrating (G), the percentage of Nkx2.1+ cells from total number of cells in three gestational ages (15, 18-19 and 22 GW) and two region - ventricular (VZ) and subventricular (SVZ) zone. (H) RT-PCR of human cortical tissue for Nkx2.1 and Lhx6 in three stages -16,18 and 19 GW; y-axis- values represent fold increase normalized to 16 GW values. (I) Western blot for transcription factors at 18 GW in the cortex (Cx) and ganglionic eminence (GE). Abbreviations: CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone. Scale bars: B: 200 µm and 50 µm, C-F: 50µm; Insets in D-F: 10µm.

Fig.4 Radial glia cell
Fig.4 Radial glia cell isolated from human fetal brain and transfected with BLBP-Cre (green) LoxP method, differentiates into oligodendrocyte progenitor labeled with O4 antibody (red). Blue- nuclear stain.

Another  part of our research is to study oligodendrocyte lineage. Studies are being done mainly in human fetal forebrain sections (15-20 gestational weeks, gw) applying immunoflorescence. Parallel experiments are done in vitro and also in age-matched mice to evaluate species-specific differences.

In RGC cultures obtained from cortical proliferative zone at midgestation we study the effect of various environmental factors, such as sonic hedgehog (SHH) and Wnt signaling, hypoxia or NMDA receptor blokers on specification and differentiation of human cortical progenitors (Radonjic et al., 2014b, Ortega et al., 2013, 2016, Bagarsawala et al 2016a,b).