Interferon regulates neural stem cell function at all ages by orchestrating mTOR and cell cycle.

Stem cells show intrinsic interferon signalling, which protects them from viral infections at all ages. In the ageing brain, interferon signalling also reduces the ability of stem cells to activate. Whether these functions are linked and at what time interferons start taking on a role in stem cell functioning is unknown. Additionally, the molecular link between interferons and activation in neural stem cells and how this relates to progenitor production is not well understood. Here we combine single-cell transcriptomics, RiboSeq and mathematical models of interferon to show that this pathway is important for proper stem cell function at all ages in mice. Interferon orchestrates cell cycle and mTOR activity to post-transcriptionally repress Sox2 and induces quiescence. The interferon response then decreases in the subsequent maturation states. Mathematical simulations indicate that this regulation is beneficial for the young and harmful for the old brain. Our study establishes molecular mechanisms of interferon in stem cells and interferons as genuine regulators of stem cell homeostasis and a potential therapeutic target to repair the ageing brain.

High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids.

Single-cell nucleosome, methylome, and transcriptome (scNMT) sequencing is a recently developed method that allows multiomics profiling of single cells. In this scNMT protocol, we describe profiling of cells from mouse brain and pancreatic organoids, using liquid handling platforms to increase throughput from 96-well to 384-well plate format. Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq3 protocol to obtain higher numbers of detected genes and genomic DNA (gDNA) CpGs per cell. We outline normalization steps to optimally distribute per-cell sequencing depth. For complete details on the use and execution of this protocol, please refer to Clark (2019), Clark et al. (2018), and Clark et al., 2018, Hagemann-Jensen et al., 2020a, Hagemann-Jensen et al., 2020b.

The crucial role of model systems in understanding the complexity of cell signaling in human neurocristopathies.

Animal models are useful to study the molecular, cellular, and morphogenetic mechanisms underlying normal and pathological development. Cell-based study models have emerged as an alternative approach to study many aspects of human embryonic development and disease. The neural crest (NC) is a transient, multipotent, and migratory embryonic cell population that generates a diverse group of cell types that arises during vertebrate development. The abnormal formation or development of the NC results in neurocristopathies (NCPs), which are characterized by a broad spectrum of functional and morphological alterations. The impaired molecular mechanisms that give rise to these multiphenotypic diseases are not entirely clear yet. This fact, added to the high incidence of these disorders in the newborn population, has led to the development of systematic approaches for their understanding. In this article, we have systematically reviewed the ways in which experimentation with different animal and cell model systems has improved our knowledge of NCPs, and how these advances might contribute to the development of better diagnostic and therapeutic tools for the treatment of these pathologies. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics Congenital Diseases > Stem Cells and Development Congenital Diseases > Molecular and Cellular Physiology Neurological Diseases > Genetics/Genomics/Epigenetics.

High throughput screening of novel AAV capsids identifies variants for transduction of adult NSCs within the subventricular zone.

The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene-therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, peptide-modified derivative of wild-type AAV9 (AAV9_A2) and peptide-modified derivative of wild-type AAV1 (AAV1_P5), both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of the promoter and dose of injected viral genomes enabled labeling of 30%-60% of the NSC compartment, which was validated by fluorescence-activated cell sorting (FACS) analyses and single-cell RNA sequencing. Importantly, transduced NSCs readily produced neurons. The present study identifies AAV variants with a high regional tropism toward the ventricular-subventricular zone (v-SVZ) with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.

The role of teratogens in neural crest development.

The neural crest (NC), discovered by Wilhelm His 150 years ago, gives rise to a multipotent migratory embryonic cell population that generates a remarkably diverse and important array of cell types during the development of the vertebrate embryo. These cells originate in the neural plate border (NPB), which is the ectoderm between the neural plate and the epidermis. They give rise to the neurons and glia of the peripheral nervous system, melanocytes, chondrocytes, smooth muscle cells, odontoblasts and neuroendocrine cells, among others. Neurocristopathies are a class of congenital diseases resulting from the abnormal induction, specification, migration, differentiation or death of NC cells (NCCs) during embryonic development and have an important medical and societal impact. In general, congenital defects affect an appreciable percentage of newborns worldwide. Some of these defects are caused by teratogens, which are agents that negatively impact the formation of tissues and organs during development. In this review, we will discuss the teratogens linked to the development of many birth defects, with a strong focus on those that specifically affect the development of the NC, thereby producing neurocristopathies. Although increasing attention is being paid to the effect of teratogens on embryonic development in general, there is a strong need to critically evaluate the specific role of these agents in NC development. Therefore, increased understanding of the role of these factors in NC development will contribute to the planning of strategies aimed at the prevention and treatment of human neurocristopathies, whose etiology was previously not considered.

Gli2 is required for the induction and migration of Xenopus laevis neural crest.

The neural crest (NC) is a multipotent migratory embryonic population that is formed during late gastrulation and gives rise to a wide array of derivatives, including cells from the peripheral nervous system (PNS), the craniofacial bones and cartilages, peripheral glial cells, and melanocyte cells, among others. In this work we analyzed the role of the Hedgehog signaling pathway effector gli2 in Xenopus NC. We provide evidence that the gli2 gene is expressed in the prospective, premigratory and migratory NC. The use of a specific morpholino against gli2 and the pharmacological specific inhibitor GANT61 in different experimental approaches allowed us to determine that gli2 is required for the induction and specification of NC cells as a transcriptional activator. Moreover, gli2 also acts by reducing apoptosis in the NC without affecting its cell proliferation status. We also demonstrated that gli2 is required cell-autonomously for NC migration, and for the formation of NC derivatives such as the craniofacial cartilages, melanocytes and the cranial ganglia. Altogether, our results showed that gli2 is a key transcriptional activator to accomplish the proper specification and development of Xenopus NC cells.

Neurocristopathies: New insights 150 years after the neural crest discovery.

The neural crest (NC) is a transient, multipotent and migratory cell population that generates an astonishingly diverse array of cell types during vertebrate development. These cells, which originate from the ectoderm in a region lateral to the neural plate in the neural fold, give rise to neurons, glia, melanocytes, chondrocytes, smooth muscle cells, odontoblasts and neuroendocrine cells, among others. Neurocristopathies (NCP) are a class of pathologies occurring in vertebrates, especially in humans that result from the abnormal specification, migration, differentiation or death of neural crest cells during embryonic development. Various pigment, skin, thyroid and hearing disorders, craniofacial and heart abnormalities, malfunctions of the digestive tract and tumors can also be considered as neurocristopathies. In this review we revisit the current classification and propose a new way to classify NCP based on the embryonic origin of the affected tissues, on recent findings regarding the molecular mechanisms that drive NC formation, and on the increased complexity of current molecular embryology techniques.

Trunk neural crest cells: formation, migration and beyond.

Neural crest cells (NCCs) are a multipotent, migratory cell population that generates an astonishingly diverse array of cell types during vertebrate development. The trunk neural crest has long been considered of particular significance. First, it has been held that the trunk neural crest has a morphogenetic role, acting to coordinate the development of the peripheral nervous system, secretory cells of the endocrine system and pigment cells of the skin. Second, the trunk neural crest additionally has skeletal potential. However, it has been demonstrated that a key role of the trunk neural crest streams is to organize the innervation of the intestine. Although trunk NCCs have a limited capacity for self-renewal, sometimes they become neural-crest-derived tumor cells and reveal the fact that that NCCs and tumor cells share the same molecular machinery. In this review we describe the routes taken by trunk NCCs and consider the signals and cues that pattern these trajectories. We also discuss recent advances in the characterization of the properties of trunk NCCs for various model organisms in order to highlight common themes. Finally, looking to the future, we discuss the need to translate the wealth of data from animal studies to the clinical area in order to develop treatments for neural crest-related human diseases.

Developmental expression and role of Kinesin Eg5 during Xenopus laevis embryogenesis.

BACKGROUND: The neural crest is a transient multipotent migratory cell population unique to vertebrates. These cells undergo an epithelial-to-mesenchymal transition and migrate extensively through the embryo. They differentiate into numerous diverse derivatives including the peripheral nervous system, melanocytes,and craniofacial cartilages. The development of the neural crest is mediated by complex interactions of multiple signals and transcription factors. The kinesin Eg5 is a plus end-directed microtubule-based motor protein that is essential for bipolar spindle formation during mitosis and meiosis, axon growth, and mammal embryonic development. RESULTS: We analyzed in detail the expression pattern of eg5 and established that it is expressed at the prospective neural fold, in the premigratory and migratory neural crest. Functional analysis revealed that in Xenopus, early embryogenesis eg5 function is required during neural crest induction, specification, and maintenance. eg5 is also required during neural crest migration and for derivatives formation. Moreover, we demonstrated a hierarchical relationship with the Indian Hedgehog signaling pathway. CONCLUSIONS: Our results show that eg5 is essential for the specification and maintenance of neural crest progenitors during Xenopus early embryogenesis rather than cell proliferation and survival.