Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

Apoptosis in Postmortal Tissues of Goat Spinal Cords and Survival of Resident Neural Progenitors.

Growing demand for therapeutic tissue repair recurrently focusses scientists' attention on critical assessment of postmortal collection of live cells, especially stem cells. Our study aimed to assess the survival of neuronal progenitors in postmortal spinal cord and their differentiation potential. Postmortal samples of spinal cords were obtained from human-sized animals (goats) at 6, 12, 24, 36, and 54 h after slaughter. Samples were studied by immunohistology, differentiation assay, Western blot and flow cytometry for the presence and location of GD2-positive neural progenitors and their susceptibility to cell death. TUNEL staining of the goat spinal cord samples over 6-54 h postmortem revealed no difference in the number of positive cells per cross-section. Many TUNEL-positive cells were located in the gray commissure around the central canal of the spinal cord; no increase in TUNEL-positive cells was recorded in either posterior or anterior horns of the gray matter where many GD2-positive neural progenitors can be found. The active caspase 3 amount as measured by Western blot at the same intervals was moderately increasing over time. Neuronal cells were enriched by magnetic separation with antibodies against CD24; among them, the GD2-positive neural progenitor subpopulation did not overlap with apoptotic cells having high pan-caspase activity. Apoptotic cell death events are relatively rare in postmortal spinal cords and are not increased in areas of the neural progenitor cell's location, within measured postmortal intervals, or among the CD24/GD2-positive cells. Data from our study suggest postmortal spinal cords as a valuable source for harvesting highly viable allogenic neural progenitor cells.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

The effects of exosomes originating from different cell sources on the differentiation of bone marrow mesenchymal stem cells into schwann cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms. MATERIALS AND METHODS: We extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways. RESULTS: BMSCs were cultured by three media: conventional (no induction), pre-induction or pre-induction + original induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The induction ability was ranked as follows: pre-induction + ODM + exosome group > pre-induction + exosome group > non-induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups. CONCLUSIONS: Our analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.

A novel adipose loss-of-function mutant in Drosophila.

To identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in Drosophila. One potential candidate isolated from this effort is the anti-lipogenic gene adipose (adp). Adp has an established role in the negative regulation of lipogenesis in the fat body of the fly and adipose tissue in mammals. While fat is key to proper development in general, adp has not been investigated during brain development. Here, we found that RNAi knockdown of adp in neuronal stem cells and neurons results in reduced brain lobe volume and sought to replicate this with a mutant fly. We generated a novel adp mutant that acts as a loss-of-function mutant based on buoyancy assay results. We found that despite a change in fat content in the body overall and a decrease in the number of larger (>5 µm) brain lipid droplets, there was no change in the brain lobe volume of mutant larvae. Overall, our work describes a novel adp mutant that can functionally replace the long-standing adp60 mutant and shows that the adp gene has no obvious involvement in brain growth.

Herpes simplex encephalitis due to a mutation in an E3 ubiquitin ligase.

Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2, in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes.

Treatment of Syringomyelia Characterized by Focal Dilatation of the Central Canal Using Mesenchymal Stem Cells and Neural Stem Cells.

BACKGROUND: Syringomyelia is a progressive chronic disease that leads to nerve pain, sensory dissociation, and dyskinesia. Symptoms often do not improve after surgery. Stem cells have been widely explored for the treatment of nervous system diseases due to their immunoregulatory and neural replacement abilities. METHODS: In this study, we used a rat model of syringomyelia characterized by focal dilatation of the central canal to explore an effective transplantation scheme and evaluate the effect of mesenchymal stem cells and induced neural stem cells for the treatment of syringomyelia. RESULTS: The results showed that cell transplantation could not only promote syrinx shrinkage but also stimulate the proliferation of ependymal cells, and the effect of this result was related to the transplantation location. These reactions appeared only when the cells were transplanted into the cavity. Additionally, we discovered that cell transplantation transformed activated microglia into the M2 phenotype. IGF1-expressing M2 microglia may play a significant role in the repair of nerve pain. CONCLUSION: Cell transplantation can promote cavity shrinkage and regulate the local inflammatory environment. Moreover, the proliferation of ependymal cells may indicate the activation of endogenous stem cells, which is important for the regeneration and repair of spinal cord injury.

Identifying tumor type and cell type-specific gene expression alterations in pediatric central nervous system tumors.

Central nervous system (CNS) tumors are the leading cause of pediatric cancer death, and these patients have an increased risk for developing secondary neoplasms. Due to the low prevalence of pediatric CNS tumors, major advances in targeted therapies have been lagging compared to other adult tumors. We collect single nuclei RNA-seq data from 84,700 nuclei of 35 pediatric CNS tumors and three non-tumoral pediatric brain tissues and characterize tumor heterogeneity and transcriptomic alterations. We distinguish cell subpopulations associated with specific tumor types including radial glial cells in ependymomas and oligodendrocyte precursor cells in astrocytomas. In tumors, we observe pathways important in neural stem cell-like populations, a cell type previously associated with therapy resistance. Lastly, we identify transcriptomic alterations among pediatric CNS tumor types compared to non-tumor tissues, while accounting for cell type effects on gene expression. Our results suggest potential tumor type and cell type-specific targets for pediatric CNS tumor treatment. Here we address current gaps in understanding single nuclei gene expression profiles of previously under-investigated tumor types and enhance current knowledge of gene expression profiles of single cells of various pediatric CNS tumors.

Human Breast Milk Exosomal miRNAs are Influenced by Premature Delivery and Affect Neurodevelopment.

SCOPE: This study investigates the exosomal microRNA (miRNA) profiles of term and preterm breast milk, including the most abundant and differentially expressed (DE) miRNAs, and their impact on neurodevelopment in infants. METHODS AND RESULTS: Mature milk is collected from the mothers of term and preterm infants. Using high-throughput sequencing and subsequent data analysis, exosomal miRNA profiles of term and preterm human breast milk (HBM) are acquired and it is found that the let-7 and miR-148 families are the most abundant miRNAs. Additionally, 23 upregulated and 15 downregulated miRNAs are identified. MiR-3168 is the most upregulated miRNA in preterm HBM exosome, exhibiting targeting activity toward multiple genes involved in the SMAD and MAPK signaling pathways and playing a crucial role in early neurodevelopment. Additionally, the effects of miR-3168 on neurodevelopment is confirmed and it is determined that it is an essential factor in the differentiation of neural stem cells (NSCs). CONCLUSION: This study demonstrates that miRNA expression in breast milk exosomes can be influenced by preterm delivery, thereby potentially impacting neurodevelopment in preterm infants.

3D hydrogel microfibers promote the differentiation of encapsulated neural stem cells and facilitate neuron protection and axon regrowth after complete transactional spinal cord injury.

Spinal cord injury (SCI) can cause permanent impairment to motor or sensory functions. Pre-cultured neural stem cell (NSC) hydrogel scaffolds have emerged as a promising approach to treat SCI by promoting anti-inflammatory effects, axon regrowth, and motor function restoration. Here, in this study, we performed a coaxial extrusion process to fabricate a core-shell hydrogel microfiber with high NSC density in the core portion. Oxidized hyaluronic acid, carboxymethyl chitosan, and matrigel blend were used as a matrix for NSC growth and to facilitate the fabrication process. During thein vitrodifferentiation culture, it was found that NSC microfibers could differentiate into neurons and astrocytes with higher efficiency compared to NSC cultured in petri dishes. Furthermore, duringin vivotransplantation, NSC microfibers were coated with polylactic acid nanosheets by electrospinning for reinforcement. The coated NSC nanofibers exhibited higher anti-inflammatory effect and lesion cavity filling rate compared with the control group. Meanwhile, more neuron- and oligodendrocyte-like cells were visualized at the lesion epicenter. Finally, axon regrowth across the whole lesion site was observed, demonstrating that the microfiber could guide renascent axon regrowth. Experiment results indicate that the NSC microfiber is a promising bioactive treatment for complete SCI treatment with superior outcomes.

Autocrine VEGF drives neural stem cell proximity to the adult hippocampus vascular niche.

The vasculature is a key component of adult brain neural stem cell (NSC) niches. In the adult mammalian hippocampus, NSCs reside in close contact with a dense capillary network. How this niche is maintained is unclear. We recently found that adult hippocampal NSCs express VEGF, a soluble factor with chemoattractive properties for vascular endothelia. Here, we show that global and NSC-specific VEGF loss led to dissociation of NSCs and their intermediate progenitor daughter cells from local vasculature. Surprisingly, though, we found no changes in local vascular density. Instead, we found that NSC-derived VEGF supports maintenance of gene expression programs in NSCs and their progeny related to cell migration and adhesion. In vitro assays revealed that blockade of VEGF receptor 2 impaired NSC motility and adhesion. Our findings suggest that NSCs maintain their own proximity to vasculature via self-stimulated VEGF signaling that supports their motility towards and/or adhesion to local blood vessels.

Reversal of Postnatal Brain Astrocytes and Ependymal Cells towards a Progenitor Phenotype in Culture.

Astrocytes and ependymal cells have been reported to be able to switch from a mature cell identity towards that of a neural stem/progenitor cell. Astrocytes are widely scattered in the brain where they exert multiple functions and are routinely targeted for in vitro and in vivo reprogramming. Ependymal cells serve more specialized functions, lining the ventricles and the central canal, and are multiciliated, epithelial-like cells that, in the spinal cord, act as bi-potent progenitors in response to injury. Here, we isolate or generate ependymal cells and post-mitotic astrocytes, respectively, from the lateral ventricles of the mouse brain and we investigate their capacity to reverse towards a progenitor-like identity in culture. Inhibition of the GSK3 and TGFß pathways facilitates the switch of mature astrocytes to Sox2-expressing, mitotic cells that generate oligodendrocytes. Although this medium allows for the expansion of quiescent NSCs, isolated from live rats by "milking of the brain", it does not fully reverse astrocytes towards the bona fide NSC identity; this is a failure correlated with a concomitant lack of neurogenic activity. Ependymal cells could be induced to enter mitosis either via exposure to neuraminidase-dependent stress or by culturing them in the presence of FGF2 and EGF. Overall, our data confirm that astrocytes and ependymal cells retain a high capacity to reverse to a progenitor identity and set up a simple and highly controlled platform for the elucidation of the molecular mechanisms that regulate this reversal.

Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains.

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance.

Neural progenitor cells within the cerebral cortex undergo a characteristic switch between symmetric self-renewing cell divisions early in development and asymmetric neurogenic divisions later. Yet, the mechanisms controlling this transition remain unclear. Previous work has shown that early but not late neural progenitor cells (NPCs) endogenously express the autism-linked transcription factor Foxp1, and both loss and gain of Foxp1 function can alter NPC activity and fate choices. Here, we show that premature loss of Foxp1 upregulates transcriptional programs regulating angiogenesis, glycolysis, and cellular responses to hypoxia. These changes coincide with a premature destabilization of HIF-1α, an elevation in HIF-1α target genes, including Vegfa in NPCs, and precocious vascular network development. In vitro experiments demonstrate that stabilization of HIF-1α in Foxp1-deficient NPCs rescues the premature differentiation phenotype and restores NPC maintenance. Our data indicate that the endogenous decline in Foxp1 expression activates the HIF-1α transcriptional program leading to changes in the tissue environment adjacent to NPCs, which, in turn, might alter their self-renewal and neurogenic capacities.

From Vessels to Neurons-The Role of Hypoxia Pathway Proteins in Embryonic Neurogenesis.

Embryonic neurogenesis can be defined as a period of prenatal development during which divisions of neural stem and progenitor cells give rise to neurons. In the central nervous system of most mammals, including humans, the majority of neocortical neurogenesis occurs before birth. It is a highly spatiotemporally organized process whose perturbations lead to cortical malformations and dysfunctions underlying neurological and psychiatric pathologies, and in which oxygen availability plays a critical role. In case of deprived oxygen conditions, known as hypoxia, the hypoxia-inducible factor (HIF) signaling pathway is activated, resulting in the selective expression of a group of genes that regulate homeostatic adaptations, including cell differentiation and survival, metabolism and angiogenesis. While a physiological degree of hypoxia is essential for proper brain development, imbalanced oxygen levels can adversely affect this process, as observed in common obstetrical pathologies such as prematurity. This review comprehensively explores and discusses the current body of knowledge regarding the role of hypoxia and the HIF pathway in embryonic neurogenesis of the mammalian cortex. Additionally, it highlights existing gaps in our understanding, presents unanswered questions, and provides avenues for future research.

Numb positively regulates Hedgehog signaling at the ciliary pocket.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

Lineage-specific changes in mitochondrial properties during neural stem cell differentiation.

Neural stem cells (NSCs) reside in discrete regions of the adult mammalian brain where they can differentiate into neurons, astrocytes, and oligodendrocytes. Several studies suggest that mitochondria have a major role in regulating NSC fate. Here, we evaluated mitochondrial properties throughout NSC differentiation and in lineage-specific cells. For this, we used the neurosphere assay model to isolate, expand, and differentiate mouse subventricular zone postnatal NSCs. We found that the levels of proteins involved in mitochondrial fusion (Mitofusin [Mfn] 1 and Mfn 2) increased, whereas proteins involved in fission (dynamin-related protein 1 [DRP1]) decreased along differentiation. Importantly, changes in mitochondrial dynamics correlated with distinct patterns of mitochondrial morphology in each lineage. Particularly, we found that the number of branched and unbranched mitochondria increased during astroglial and neuronal differentiation, whereas the area occupied by mitochondrial structures significantly reduced with oligodendrocyte maturation. In addition, comparing the three lineages, neurons revealed to be the most energetically flexible, whereas astrocytes presented the highest ATP content. Our work identified putative mitochondrial targets to enhance lineage-directed differentiation of mouse subventricular zone-derived NSCs.

The pivotal role of irradiation-induced apoptosis in the pathogenesis and therapy of medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is a rare primitive neuroectodermal tumors originating from the cerebellum. MB is the most common malignant primary brain tumor of childhood. MB originates from neural precursor cells in distinctive regions of the rhombic lip, and their maturation occurs in the cerebellum or the brain stem during embryonal development. Also, apoptosis is a programmed cell death associated with numerous physiological as well as pathological regulations. RECENT FINDINGS: Irradiation (IR)-induce apoptosis triggers cell death, with or without intervening mitosis within a few hours of IR and these share different morphologic alteration such as, loss of normal nuclear structure as well as degradation of DNA. Moreover, MB is strikingly sensitive to DNA-damaging therapies and the role of apoptosis a key treatment modality. Furthermore, in MB, the apoptotic pathways are made up of several triggers, modulators, as well as effectors. Notably, IR-induced apoptotic mechanisms in MB therapy are very complex and they either induce radiosensitivity or inhibit radioresistance leading to potential effective treatment strategies for MB. CONCLUSION: This review explicitly explores the pivotal roles of IR-induced apoptosis in the pathogenesis and therapy of MB.

Dual and opposing roles for the kinesin-2 motor, KIF17, in Hedgehog-dependent cerebellar development.

While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion partially phenocopies Kif17 germline mutants. Unexpectedly, CGNP-specific Kif17 deletion results in the opposite phenotype-increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together, these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPs.

Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development.

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.