Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly

Primary microcephaly and megalencephaly are severe brain malformations defined by reduced and increased brain size, respectively. Whether these two pathologies arise from related alterations at the molecular level is unclear. Microcephaly has been largely associated with centrosomal defects, leading to cell death. Here, we investigate the consequences of WDR81 loss of function, which causes severe microcephaly in patients. We show that WDR81 regulates endosomal trafficking of EGFR and that loss of function leads to reduced MAP kinase pathway activation. Mouse radial glial progenitor cells knocked-out for WDR81 exhibit reduced proliferation rate, subsequently leading to reduced brain size. These proliferation defects are rescued in vivo by expressing a megalencephaly-causing mutant form of Cyclin D2. Our results identify the endosomal machinery as an important regulator of proliferation rates and brain growth, demonstrating that microcephaly and megalencephaly can be caused by opposite effects on the proliferation rate of radial glial progenitors.

Transcriptional network orchestrating regional patterning of cortical progenitors

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer–gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

Calcium Signaling in the Cerebellar Radial Glia and Its Association with Morphological Changes during Zebrafish Development

Radial glial cells are a distinct non-neuronal cell type that, during development, span the entire width of the brain walls of the ventricular system. They play a central role in the origin and placement of neurons, since their processes form structural scaffolds that guide and facilitate neuronal migration. Furthermore, glutamatergic signaling in the radial glia of the adult cerebellum (i.e., Bergmann glia), is crucial for precise motor coordination. Radial glial cells exhibit spontaneous calcium activity and functional coupling spread calcium waves. However, the origin of calcium activity in relation to the ontogeny of cerebellar radial glia has not been widely explored, and many questions remain unanswered regarding the role of radial glia in brain development in health and disease. In this study we used a combination of whole mount immunofluorescence and calcium imaging in transgenic (gfap-GCaMP6s) zebrafish to determine how development of calcium activity is related to morphological changes of the cerebellum. We found that the morphological changes in cerebellar radial glia are quite dynamic; the cells are remarkably larger and more elaborate in their soma size, process length and numbers after 7 days post fertilization. Spontaneous calcium events were scarce during the first 3 days of development and calcium waves appeared on day 5, which is associated with the onset of more complex morphologies of radial glia. Blockage of gap junction coupling inhibited the propagation of calcium waves, but not basal local calcium activity. This work establishes crucial clues in radial glia organization, morphology and calcium signaling during development and provides insight into its role in complex behavioral paradigms.

Gliogenic Potential of Single Pallial Radial Glial Cells in Lower Cortical Layers

During embryonic development, progenitor cells are progressively restricted in their potential to generate different neural cells. A specific progenitor cell type, the radial glial cells, divides symmetrically and then asymmetrically to produce neurons, astrocytes, oligodendrocytes, and NG2-glia in the cerebral cortex. However, the potential of individual progenitors to form glial lineages remains poorly understood. To further investigate the cell progeny of single pallial GFAP-expressing progenitors, we used the in vivo genetic lineage-tracing method, the UbC-(GFAP-PB)-StarTrack. After targeting those progenitors in embryonic mice brains, we tracked their adult glial progeny in lower cortical layers. Clonal analyses revealed the presence of clones containing sibling cells of either a glial cell type (uniform clones) or two different glial cell types (mixed clones). Further, the clonal size and rostro-caudal cell dispersion of sibling cells differed depending on the cell type. We concluded that pallial E14 neural progenitors are a heterogeneous cell population with respect to which glial cell type they produce, as well as the clonal size of their cell progeny.

Inhibited maturation of astrocytes caused by maternal n-3 polyunsaturated fatty acid intake deficiency hinders the development of brain glial cells in neonatal rats

The brain is rich in long chain polyunsaturated fatty acids (PUFAs), which play an essential role in its development and functions. Here we examined the impact of maternal n-3 PUFA intake deficiency during gestation and lactation on the development of glial cells in the pup’s developing cerebral cortex. In addition, using myelination as indicator and the anti-myelin basic protein (MBP) as measurement to establish the relationship between the number of glial fibrillary acidic protein (GFAP)-positive cells and the development of oligodendrocytes, we determined the myelination state of the somatosensory cortex at day 14 postnatal. Rat dams were fed either a control (Cont) or an n-3 PUFA-deficient (Def) diet for 60 days (acclimatisation :14 days; gestation: 21 days; lactation:21 days). Pups lactated from dams throughout the experiment. The distribution pattern of astrocytes in pups on day 7 postnatal was immunohistochemically analysed using GFAP and brain lipid binding protein (BLBP) as markers for mature astrocytes and astrocyte-specific radial glial cells, respectively. It was observed that, when compared with Cont pups, GFAP-positive cells decreased, BLBP-positive cells increased and myelinated structures were sparser in the somatosensory cortices of Def pups. In the open field test on day 21 postnatal, behavioural parameters did not differ between groups. Our results indicated that inhibited maturation of astrocytes caused by maternal n-3 PUFA deficiency hindered the development of brain glial cells of neonatal rats and hence, maternal n-3 PUFA intake during the gestation and lactation periods may have been crucial for the brain cell composition of pups.

Spatiotemporal transcriptome at single-cell resolution reveals key radial glial cell population in axolotl telencephalon development and regeneration

Brain regeneration requires a precise coordination of complex responses in a time- and region-specific manner. Identifying key cell types and molecules that direct brain regeneration would provide potential targets for the advance of regenerative medicine. However, progress in the field has been hampered largely due to very limited regeneration capacity of the mammalian brain and understanding of the regeneration process at both cellular and molecular level. Here, using axolotl brain with astonishing regeneration ability upon injury, and the Stereo-seq (SpaTial Enhanced REsolution Omics-sequencing), we reconstruct the first architecture of axolotl telencephalon with gene expression profiling at single-cell resolution, and fine cell dynamics maps throughout development and regeneration. Intriguingly, we discover a marked heterogeneity of radial glial cell (RGC) types with distinct behaviors. Of note, one subtype of RGCs is activated since early regeneration stages and proliferates while other RGCs remain dormant. Such RGC subtype appears to be the major cell population involved in early wound healing response and gradually covers the injured area before presumably transformed into the lost neurons. Altogether, our work systematically decodes the complex cellular and molecular dynamics of axolotl telencephalon in development and regeneration, laying the foundation for studying the regulatory mechanism of brain regeneration in future.

Trp53 ablation fails to prevent microcephaly in mouse pallium with impaired minor intron splicing

Minor spliceosome inhibition due to mutations in RNU4ATAC are linked to primary microcephaly. Ablation of Rnu11, a minor spliceosome snRNA, inhibits the minor spliceosome in the developing mouse pallium, causing microcephaly. There, cell cycle defects and p53-mediated apoptosis in response to DNA damage resulted in loss of radial glial cells (RGCs), underpinning microcephaly. Here, we ablated Trp53 to block cell death in the Rnu11 cKO mice. We report that Trp53 ablation failed to prevent microcephaly in these double knockout (dKO) mice. We show that the transcriptome of the dKO pallium was closer to the control compared to the Rnu11 cKO. We find aberrant minor intron splicing in MIGs involved in cell cycle regulation, resulting in more severely impaired mitotic progression and cell cycle lengthening of RGCs in the dKO that was detected earlier than the Rnu11 cKO. Furthermore, we discover a potential role of p53 in causing DNA damage in the developing pallium, as detection of γH2aX+ was delayed in the dKO. Thus, we postulate that microcephaly in minor spliceosome-related diseases is primarily caused by cell cycle defects.

Review and Hypothesis: A Potential Common Link Between Glial Cells, Calcium Changes, Modulation of Synaptic Transmission, Spreading Depression, Migraine, and Epilepsy—H+

There is significant evidence to support the notion that glial cells can modulate the strength of synaptic connections between nerve cells, and it has further been suggested that alterations in intracellular calcium are likely to play a key role in this process. However, the molecular mechanism(s) by which glial cells modulate neuronal signaling remains contentiously debated. Recent experiments have suggested that alterations in extracellular H+ efflux initiated by extracellular ATP may play a key role in the modulation of synaptic strength by radial glial cells in the retina and astrocytes throughout the brain. ATP-elicited alterations in H+ flux from radial glial cells were first detected from Müller cells enzymatically dissociated from the retina of tiger salamander using self-referencing H+-selective microelectrodes. The ATP-elicited alteration in H+ efflux was further found to be highly evolutionarily conserved, extending to Müller cells isolated from species as diverse as lamprey, skate, rat, mouse, monkey and human. More recently, self-referencing H+-selective electrodes have been used to detect ATP-elicited alterations in H+ efflux around individual mammalian astrocytes from the cortex and hippocampus. Tied to increases in intracellular calcium, these ATP-induced extracellular acidifications are well-positioned to be key mediators of synaptic modulation. In this article, we examine the evidence supporting H+ as a key modulator of neurotransmission, review data showing that extracellular ATP elicits an increase in H+ efflux from glial cells, and describe the potential signal transduction pathways involved in glial cell—mediated H+ efflux. We then examine the potential role that extracellular H+ released by glia might play in regulating synaptic transmission within the vertebrate retina, and then expand the focus to discuss potential roles in spreading depression, migraine, epilepsy, and alterations in brain rhythms, and suggest that alterations in extracellular H+ may be a unifying feature linking these disparate phenomena.