Zbtb20 modulates the sequential generation of neuronal layers in developing cortex
The mammalian neocortex (Ncx), in which neurons are arranged radially in six layers and tangentially in numerous functional domains, is a recent acquisition in brain evolution. During development, the majority of cortical glutamatergic neurons are generated by radial glial cells (RGCs) in the germinative ventricular (VZ) and subventricular (SVZ) zone of the dorsolateral pallium. Generation of neuronal sets with a layer-specific identity depends on an intrinsically encoded genetic program and environmental cues acting during the S-phase of the mitotic cycle [1]. Neurons with different fates are produced according to an “inside-first outside-last” schedule: first, lower layer (LL) neurons (L6/L5), followed by generation of the upper layer (UL) neurons (L4/L3/L2). During mouse development, the layer specific neuronal subtypes are generated throughout embryonic (E) stages E10.5 – E17.5 in partially overlapping time windows with a peak for generation at E11.5 for L1, E12.5 (L6), E13.5 (L5), E14.5 (L4) and E16.5-E17.5 (L3-L2) [2–5]. The birthdate of cortical neurons is directly also related to their projection identity. Thus, while the early born L6 and L5 neurons extend outside the brain thalamocortical (TCA) and corticospinal motor neuron (CSMN) projections, the late-born UL neurons make interhemispheric (callosal) projections inside the brain [6]. Increasing recent evidence support the view that the precise temporal programs for production of LL and UL neuronal fates relies on intrinsic mechanisms in early and late progenitors, respectively, characterized by specific combinatorial expression of TFs at distinct developmental time points [7–13]. For instance, suppression of the expression of Foxg1 at E10.5 is required to make a switch from generation of reelin-positive Cajal-Retzius cells, located in the marginal zone (MZ) of the cortex, to the production of neuronal subsets located in cortical plate (CP) [14]. Furthermore, while Fezf2 and Otx1 expression in apical VZ progenitors controls the fate specification of the LL neurons [15, 16], the expression of Svet1, Cux1 and Cux2 during later stages of neurogenesis in SVZ progenitors seems to specify UL neuronal fate [17–20]. The expression of TFs in postmitotic CP neurons may regulate through feedback signalling mechanism the progenitor progeny in the germinative zone [21] or the fate of the postmitotic neurons [7–13].
Neurons with distinct morphology, connectivity, neurotransmitter usage and function are tangentially organized in numerous functional domains, implicating that mechanisms of layer and area formation are interrelated. According to the current view, cortical arealization is presaged by encoded positional information (“protomap”) through graded expression of sets of TFs along the anteroposterior and mediolateral axis in the two germinative zones of the neocortex [5, 22]. Disruption of the graded expression of such TFs in VZ/SVZ leads to severe defects in the areal size and location in the Ncx [23–25]. As recently shown, affecting the intrinsic genetic mechanisms encoded by the graded expression of TF Pax6 in cortical progenitors results in an altered size of cortical somatosensory (SS) area and in parallel alterations in the sensory thalamus involving selective death of neurons in particular thalamic nuclei. Consequently, a new type of “top-down plasticity” driven by competition-mediated axon elimination and neuronal apoptosis re-patterns the sensory thalamus [26].
In a microarray screen aimed to find out genes with graded expression in the developing cortex, we identified TF Zbtb20 as a gene showing caudal-high to rostral-low expression gradient in VZ of E16.5 cortex, and subsequently maintains a restricted high expression in the adult hippocampus (Hi) [27]. The gene Zbtb20 (also named DPZF [28], HOF [29] or ZNF288) encodes a TF, belonging to the POK-family of BTB zinc finger transcriptional repressors, implicated in developmental processes and cancer [30]. Zbtb20 has been localized exclusively in immature post-mitotic neurons in Hi and migrating granule cell precursors of DG [29]. Previous research, using both gain-of-function (GOF) [31–33] and loss-of-function (LOF) [34, 35] approaches, has described the important role of TF Zbtb20 in specifying the medial pallium, the anlage of the Hi formation.
Recent studies have implicated Zbtb20 mutations in human neurodevelopmental syndromes associated with behavioral abnormalities, including intellectual disability [36], Primrose syndrome [37], autism [38], and schizophrenia [39]. The reported alterations in brain morphology in these disorders suggest possible cortical involvement beyond the hippocampus. However, no data are presently available on the involvement of Zbtb20 in neocortical morphogenesis.
In this study, we present first evidence that TF Zbtb20 exerts a dynamic expression in the germinative zones of the cortex (pallium), marks specifically the uppermost L3-L2 cortical neurons and exerts a crucial control in the timely generation of distinct neuronal fates throughout cortical neurogenesis.