PDK1 Regulates the Lengthening of G1 Phase to Balance RGC Proliferation and Differentiation during Cortical Neurogenesis.

During cortical development, the balance between progenitor self-renewal and neurogenesis is critical for determining the size/morphology of the cortex. A fundamental feature of the developing cortex is an increase in the length of G1 phase in RGCs over the course of neurogenesis, which is a key determinant of progenitor fate choice. How the G1 length is temporally regulated remains unclear. Here, Pdk1, a member of the AGC kinase family, was conditionally disrupted by crossing an Emx1-Cre mouse line with a Pdk1fl/fl line. The loss of Pdk1 led to a shorter cell cycle accompanied by increased RGC proliferation specifically at late rather than early/middle neurogenic stages, which was attributed to impaired lengthening of G1 phase. Coincidently, apical-to-basal interkinetic nuclear migration was accelerated in Pdk1 cKO cortices. Consequently, we detected an increased neuronal output at P0. We further showed the significant upregulation of the cell cycle regulator cyclin D1 and its activator Myc in the cKO cortices relative to those of control animals. Overall, we have identified a novel role for PDK1 in cortical neurogenesis. PDK1 functions as an upstream regulator of the Myc-cyclin D1 pathway to control the lengthening of G1 phase and the balance between RGC proliferation and differentiation.

Foxg1 Regulates the Postnatal Development of Cortical Interneurons.

Abnormalities in cortical interneurons are closely associated with neurological diseases. Most patients with Foxg1 syndrome experience seizures, suggesting a possible role of Foxg1 in the cortical interneuron development. Here, by conditional deletion of Foxg1, which was achieved by crossing Foxg1fl/fl with the Gad2-CreER line, we found the postnatal distributions of somatostatin-, calretinin-, and neuropeptide Y-positive interneurons in the cortex were impaired. Further investigations revealed an enhanced dendritic complexity and decreased migration capacity of Foxg1-deficient interneurons, accompanied by remarkable downregulation of Dlx1 and CXCR4. Overexpression of Dlx1 or knock down its downstream Pak3 rescued the differentiation detects, demonstrated that Foxg1 functioned upstream of Dlx1-Pak3 signal pathway to regulate the postnatal development of cortical interneurons. Due to the imbalanced neural circuit, Foxg1 mutants showed increased seizure susceptibility. These findings will improve our understanding of the postnatal development of interneurons and help to elucidate the mechanisms underlying seizure in patients carrying Foxg1 mutations.

FOXG1 drives transcriptomic networks to specify principal neuron subtypes during the development of the medial pallium.

The medial pallium (MP) is the major forebrain region underlying learning and memory, spatial navigation, and emotion; however, the mechanisms underlying the specification of its principal neuron subtypes remain largely unexplored. Here, by postmitotic deletion of FOXG1 (a transcription factor linked to autism spectrum disorders and FOXG1 syndrome) and single-cell RNA sequencing of E17.5 MP in mice, we found that FOXG1 controls the specification of upper-layer retrosplenial cortical pyramidal neurons [RSC-PyNs (UL)], subiculum PyNs (SubC-PyNs), CA1-PyNs, CA3-PyNs, and dentate gyrus granule cells (DG-GCs) in the MP. We uncovered subtype-specific and subtype-shared FOXG1-regulated transcriptomic networks orchestrating MP neuron specification. We further demonstrated that FOXG1 transcriptionally represses Zbtb20, Prox1, and Epha4 to prevent CA3-PyN and DG-GC identities during the specification of RSC-PyNs (UL) and SubC-PyNs; FOXG1 directly activates Nr4a2 to promote SubC-PyN identity. We showed that TBR1, controlled by FOXG1 during CA1-PyN specification, was down-regulated. Thus, our study illuminates MP principal neuron subtype specification and related neuropathogenesis.

Expression pattern of NLRC5 in the postnatal mouse brain.

Nucleotide oligomerization domain-like receptors (NLRs), belonging to a large family of pattern recognition receptors, participate in the host's first line of defense against invading pathogens. Caspase recruitment domain containing 5 (NLRC5), the largest member in the NLR family, is demonstrated to be involved in the innate immune response and inflammatory diseases far and wide. Recent studies report that NLRC5 is associated with some central nervous system (CNS) diseases. Besides, NLRC5 is a mastery regulator for the expression of MHC class I both in the immune system and the CNS, while MHC class I is expressed and exerts its function in the brain. Therefore, it is necessary to investigate the expression pattern of NLRC5 in the developing and adult CNS. In our study, postnatal brain sections of C57BL/6 J mice are analyzed for the expression of NLRC5 protein by immunofluorescence. In the postnatal stages of developing telencephalon, NLRC5 exhibits a spatial and temporal expression pattern. NLRC5 is time-specifically expressed in subfields of hippocampus and different layers of prefrontal cortex. Moreover, it is shown that NLRC5 is highly cell type specific. It can be expressed in large quantities by neurons and microglia, but rarely expressed by astrocytes. Taken together, our research is important for further understanding the biological characteristics of NLRC5 and its function in the CNS.

FOXG1 sequentially orchestrates subtype specification of postmitotic cortical projection neurons.

The mammalian neocortex is a highly organized six-layered structure with four major cortical neuron subtypes: corticothalamic projection neurons (CThPNs), subcerebral projection neurons (SCPNs), deep callosal projection neurons (CPNs), and superficial CPNs. Here, careful examination of multiple conditional knockout model mouse lines showed that the transcription factor FOXG1 functions as a master regulator of postmitotic cortical neuron specification and found that mice lacking functional FOXG1 exhibited projection deficits. Before embryonic day 14.5 (E14.5), FOXG1 enforces deep CPN identity in postmitotic neurons by activating Satb2 but repressing Bcl11b and Tbr1. After E14.5, FOXG1 exerts specification functions in distinct layers via differential regulation of Bcl11b and Tbr1, including specification of superficial versus deep CPNs and enforcement of CThPN identity. FOXG1 controls CThPN versus SCPN fate by fine-tuning Fezf2 levels through diverse interactions with multiple SOX family proteins. Thus, our study supports a developmental model to explain the postmitotic specification of four cortical projection neuron subtypes and sheds light on neuropathogenesis.

FOXG1 Directly Suppresses Wnt5a During the Development of the Hippocampus.

The Wnt signaling pathway plays key roles in various developmental processes. Wnt5a, which activates the non-canonical pathway, has been shown to be particularly important for axon guidance and outgrowth as well as dendrite morphogenesis. However, the mechanism underlying the regulation of Wnt5a remains unclear. Here, through conditional disruption of Foxg1 in hippocampal progenitors and postmitotic neurons achieved by crossing Foxg1fl/fl with Emx1-Cre and Nex-Cre, respectively, we found that Wnt5a rather than Wnt3a/Wnt2b was markedly upregulated. Overexpression of Foxg1 had the opposite effects along with decreased dendritic complexity and reduced mossy fibers in the hippocampus. We further demonstrated that FOXG1 directly repressed Wnt5a by binding to its promoter and one enhancer site. These results expand our knowledge of the interaction between Foxg1 and Wnt signaling and help elucidate the mechanisms underlying hippocampal development.

Brpf1 Haploinsufficiency Impairs Dendritic Arborization and Spine Formation, Leading to Cognitive Deficits.

Haploinsufficiency of the bromodomain and PHD finger-containing protein 1 (BRPF1) gene causes intellectual disability (ID), which is characterized by impaired intellectual and cognitive function; however, the neurological basis for ID and the neurological function of BRPF1 dosage in the brain remain unclear. Here, by crossing Emx1-cre mice with Brpf1fl/fl mice, we generated Brpf1 heterozygous mice to model BRPF1-related ID. Brpf1 heterozygotes showed reduced dendritic complexity in both hippocampal granule cells and cortical pyramidal neurons, accompanied by reduced spine density and altered spine and synapse morphology. An in vitro study of Brpf1 haploinsufficiency also demonstrated decreased frequency and amplitude of miniature EPSCs that may subsequently contribute to abnormal behaviors, including decreased anxiety levels and defective learning and memory. Our results demonstrate a critical role for Brpf1 dosage in neuron dendrite arborization, spine morphogenesis and behavior and provide insight into the pathogenesis of BRPF1-related ID.

Fstl1 is involved in the regulation of radial glial scaffold development.

BACKGROUND: Radial glial cells (RGCs), the instructive scaffolds for neuronal migration, are well characterized by their unique morphology and polarization; these cells extend elongated basal processes to the pial basement membrane (BM) and parallel to one another. However, little is known about the mechanisms that underlie the developmental regulation and maintenance of this unique morphology. RESULTS: Here, by crossing Fstl1 (fl/fl) mice with an EIIa-Cre line, we identified a new role for the secreted glycoprotein Follistatin like-1 (FSTL1). The ablation of Fstl1 in both of its cortical expression domains, the ventricular zone (VZ) and the pia mater, resulted in RGC morphologic disruption; basal processes were not parallel to each other, and endfeet exhibited greater density and branching. However, Fstl1 deletion in only the VZ in the Emx1 (IREScre); Fstl1 (fl/fl) line did not affect RGC morphology, indicating that FSTL1 derived from the pia mater might be more important for RGC morphology. In addition, upper-layer projection neurons, not deeper-layer projection neurons, failed to reach their appropriate positions. We also found that BMP, AKT/PKB, Cdc42, GSK3ß, integrin and reelin signals, which have previously been reported to regulate RGC development, were unchanged, indicating that Fstl1 may function through a unique mechanism. CONCLUSIONS: In the present study, we identified a new role for FSTL1 in the development of radial glial scaffolds and the neuronal migration of upper-layer projection neurons. Our findings will improve understanding of the regulation of RGC development and neuronal migration.