Structural and Functional Aspects of the Neurodevelopmental Gene NR2F1: From Animal Models to Human Pathology

Abstract

The assembly and maturation of the mammalian brain result from an intricate cascade of highly coordinated developmental events, such as cell proliferation, migration, and differentiation. Any impairment of this delicate multi-factorial process can lead to complex neurodevelopmental diseases, sharing common pathogenic mechanisms and molecular pathways resulting in multiple clinical signs. A recently described monogenic neurodevelopmental syndrome named Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS) is caused by NR2F1 haploinsufficiency. The NR2F1 gene, coding for a transcriptional regulator belonging to the steroid/thyroid hormone receptor superfamily, is known to play key roles in several brain developmental processes, from proliferation and differentiation of neural progenitors to migration and identity acquisition of neocortical neurons. In a clinical context, the disruption of these cellular processes could underlie the pathogenesis of several symptoms affecting BBSOAS patients, such as intellectual disability, visual impairment, epilepsy, and autistic traits. In this review, we will introduce NR2F1 protein structure, molecular functioning, and expression profile in the developing mouse brain. Then, we will focus on Nr2f1 several functions during cortical development, from neocortical area and cell-type specification to maturation of network activity, hippocampal development governing learning behaviors, assembly of the visual system, and finally establishment of cortico-spinal descending tracts regulating motor execution. Whenever possible, we will link experimental findings in animal or cellular models to corresponding features of the human pathology. Finally, we will highlight some of the unresolved questions on the diverse functions played by Nr2f1 during brain development, in order to propose future research directions. All in all, we believe that understanding BBSOAS mechanisms will contribute to further unveiling pathophysiological mechanisms shared by several neurodevelopmental disorders and eventually lead to effective treatments.

Keywords: BBSOAS; NR2F1; cortical development; mouse models; neurodevelopmental disease.

Figure 1

NR2F1 structure and molecular mechanisms of gene expression regulation. (A) Human NR2F1 linear structure showing conserved protein domains. The activation function (AF) 1 and 2, the DNA-binding domain (DBD) and ligand-binding domain (LBD) are depicted as circles and boxes, respectively. (B) Schematic model of an NR2F1 dimer, based upon predictive homology obtained by using NR2F2 structure as a template (96% aminoacidic sequence homology with NR2F1; Kruse et al., 2008). In the scheme, the LBD mediates the dimerization, whereas the DBD, consisting of two highly conserved zinc finger domains, are implicated in the interaction with the double DNA helix. Between the two domains, an undefined region depicted as a loose string, which does not contain secondary structures, has not been resolved to date. (C–F) Different regulatory mechanisms carried out by Nr2f1. The nuclear receptor can act both as a direct inhibitor (C), as a competitor by binding to the regulatory sequence of target genes, and/or by sequestering other nuclear receptors as heterodimers (D), or as an activator, either directly (E) or indirectly (F), by interacting with other factors, such as Sp1, to induce transcription. AF-1/2, activation function domain 1 and 2; DBD, DNA-binding domain; DR1, direct repeat sites (spaced by one nucleotide); N-CoR, nuclear receptor co-repressor; NR, nuclear receptor; RXR, retinoic X receptor; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor; Sp1, transcription factor specificity protein 1. Modified from Tang et al. (2015) and Bertacchi et al. (2019b).

Figure 2

Nr2f1-mediated control of cellular and morphological dynamics during early corticogenesis. Schematic illustration of Nr2f1 antero-low to postero-high expression gradient (blue color code, brain scheme on the left) and latero-high to medial-low gradient (transversal sections on the right) in the developing neocortex. Nr2f1 expression in neural progenitors (NPs) spans from the ventricular zone (VZ) in apical radial glia cells (aRGs) to sub-ventricular zone (SVZ) in intermediate progenitors (IPs) and basal RGs (bRGs). RGs produce neurons directly or indirectly, via IPs, newly generated neurons migrating towards the intermediate zone (IZ) and then forming distinct layers in the cortical plate (CP). Upon Nr2f1 loss (null mutants), the posterior NP pool expands leading to an occipital enlargement, reminiscent of megalencephaly. The NP pool expansion is caused by cell cycle acceleration (round arrows), increased symmetrical divisions and self-renewal, and delayed neurogenesis causing an early decrease of IPs and of migrating and differentiating neurons in IZ and CP. At later stages, IPs, bRGs and neurons are produced at a high rate, leading to the formation of a thicker posterior CP in mutant embryos compared to wild type. The morphological consequences of Nr2f1 loss are an early lateral expansion followed by late radial expansion (gray arrows) of cortical hemispheres. At the molecular level, Nr2f1 orchestrates NP cell cycle progression and neural differentiation by repressing Pax6 and Dct and thus cell cycling progression, while activating P21-mediated cell cycle exit to promote terminal neural differentiation. aRG, apical radial glia; bRG, basal radial glia; CP, cortical plate; IZ, intermediate zone; SVZ, sub-ventricular zone; VZ, ventricular zone.

Figure 3

Nr2f1 graded expression drives cortical arealization during embryonic development. (A) Nr2f1 graded expression (blue color code) in the telencephalon starts at embryonic day (E) 9.5 posteriorly and then spreads forming a diffuse gradient from high postero-lateral to low antero-medial levels in the post-natal (P) 0 cortex. From the onset of area refinement (here represented at P7), Nr2f1 high expression is maintained in the primary sensory areas (S1, V1 and A1), but almost absent from secondary sensory areas and from the frontal motor area (M1). (B) Graphic representing the different steps of the arealization process. Starting from E8.0, several morphogens (SHH, WNTs, BMPs, SFRP2, EGFs, and FGF8 among others) are secreted from distinctly located patterning centers and diffuse along gradients in the developing neocortex, in turns driving the graded expression of transcription factors (TFs) in cortical progenitor cells. The strong caudal expression of Emx2 and Nr2f1 promotes the specification of sensory areas, while rostral high expression of Pax6 and Sp8 drives the specification of motor identity. This “protomap” is then refined with the arrival of thalamic axons (TCA innervation) conveying external sensory information. (C) Schematic of M1 (pink), S1 (green), V1 (purple) and A1 (yellow) areas in wild-type brains and in different mouse models of Nr2f1 downregulation (constitutive knock-out -KO- and conditional one -cKO-) and upregulation (knock-in -KI-). The right hemisphere depicts Nr2f1 expression, while the left one schematizes the position and size of distinct areas upon genetic manipulation. To note, changes in size and extension of the functional areas are not associated with significant changes in overall cortex volume, with the only exception of caudal megalencephaly in null animals (see Figure 2). A1, primary auditory area; M1, primary motor area; S1, primary somatosensory area; V1, primary visual area.

Figure 4

Triple Nr2f1-mediated control of neocortical mapping. The Nr2f1 gradient in the developing neocortex (blue color code) is key for a regional-specific control of three fundamental processes of corticogenesis: cell proliferation (A), cell migration (B) and neuronal identity acquisition (C). (A) In progenitor cells, Nr2f1 regulates cell cycle progression and the balance between self-renewal and differentiation (neurogenesis), ultimately controlling the number of newborn neurons locally produced along neocortical axes. (B) In newborn neurons, Nr2f1 controls the rate and efficiency of radial migration , via the regulation of Rnd2 expression. (C) Post-mitotically, Nr2f1 leads the establishment and consolidation of mature neuron identity. Being expressed in both progenitors and neurons allows Nr2f1 to connect all these distinct cellular processes, ultimately controlling the position and size of neocortical areas. CP, cortical plate; IZ, intermediate zone; SVZ, sub-ventricular zone; VZ, ventricular zone.

Figure 5

Nr2f1 controls the bioelectric and cytostructural nature of layer V cortical neurons. (A) Schematic representation of a layer V pyramidal neuron in a physiological context. A specific asset of ion channels is displayed on the cell membrane (magnification in the black dashed box) and determines cell electrophysiological properties (i.e., resting membrane potential, sag current, etc.) and proper network synchronization. The firing pattern of five neurons -N1 to N5- is schematized in the lower scheme, in the form of gray squares (representing firing events) distributed along lines (indicating measurement time during recording). Structural features of dendrite complexity and axon initial segment (AIS; magnified in the red dashed box) are also influenced by the cellular bioelectric state. (B) Summary of the main defects observed upon loss of Nr2f1. Several ion channels are downregulated in mutant neurons (inset in the black dashed box), resulting in increased intrinsic excitability. Furthermore, distinct neurons lose their ability to efficiently fire action potentials in a synchronized way (lower scheme), suggesting that the overall network synchronization is affected. At the morphological level, the complexity of the basal dendrites is drastically diminished upon Nr2f1 loss, and the AIS is reduced in size and misplaced closer to the soma (magnified in the red dashed box). Modified from Del Pino et al. (2020). LV, layer V.

Figure 6

Layer V pyramidal neuron subtype specification is under the control of Nr2f1 cortical expression. In wild-type mice (A), subcortical projecting neurons of layer V target distinct brain regions depending on their area localization. Layer V neurons from the fronto-motor areas (F/M, orange) mainly project to the spinal cord (SC) with very few axons targeting the pontine nuclei (PN), whereas layer V neurons from S1 (green) mainly target the pontine nuclei with fewer projections towards the SC. In conditional cortical-specific Nr2f1 mutants (Emx1-cKO) (B), F/M axons are incorrectly wired and stop at the level of the cerebral peduncle (CP), resulting in a depletion of corticospinal connections upon Nr2f1 loss. Layer V axons from the motorized S1 (mS1) still project to the PN (green/orange dashed lines), but not to the SC, which is instead aberrantly innervated by layer VI mS1 neurons (blue/orange dashed line). A1, primary auditory area; CP, cerebral peduncle; F/M, fronto-motor area; LV, layer V; LVI, layer VI; mS1, motorized primary somatosensory area; PN, pontine nucleus; S1, primary somatosensory area; SC, spinal cord; V1, primary visual area.

Figure 7

Nr2f1-dependent development of the peripheral and central visual system in mouse. (A) Overview of the phenotypes observed in several structures of the mouse visual system upon Nr2f1 loss, comprising the neural retina (NR), the optic disc (OD), the optic nerve (ON), and distinct central structures, such as the dorsolateral geniculate nucleus (dLGN) and the primary visual area (V1). (B) Nr2f1 loss causes a molecular shift of the Pax6+ NR domain towards the Pax2+ optic stalk (OS)/ON domain, resulting in aberrant positioning of the OD border and axonal misguidance of retinal ganglion cell (RGC) axons exiting the developing eyeball. (C) Nr2f1 haploinsufficient (HET) or depleted (KO) mice show decreased size of the ON compared to wild-type (WT) animals, resembling ON atrophy reported in BBSOAS patients. Upon Nr2f1 loss, a population of Sox2+ astrocytes with inflamed morphology outnumbers oligodendrocyte progenitors (OPCs), resulting in ON inflammation and hypomyelination. While the myelination defect can be rescued by Miconazole treatment, the high proportion of reactive astrocytes and consequent gliosis and the general ON atrophy are not reverted by pro-myelinating chemical drugs. (D) Reduced axonal innervation and connectivity (in red) from the NR to the atrophic dLGN (in blue) in Nr2f1-deficient mice. (E) Atrophic V1 (blue) and impaired maturation of secondary associative visual cortices (visual high order cortex -V^HO-; gray) due to arealization defects upon Nr2f1 loss. A1, primary auditory area; dLGN, dorsolateral geniculate nucleus; F/M, frontal/motor area; NR, neural retina; OD, optic disc; ON, optic nerve; OPC, oligodendrocyte precursor cell; OS, optic stalk; S1, primary somatosensory area; V1, primary visual area; V^HO, high-order visual associative secondary areas; vLGN, ventrolateral geniculate nucleus.