Tlx3 mediates neuronal differentiation and proper condensation of the developing trigeminal ganglion.

The trigeminal ganglion, the largest of the vertebrate cranial ganglia, is comprised of sensory neurons that relay sensations of pain, touch, and temperature to the brain. These neurons are derived from two embryonic cell types, the neural crest and ectodermal placodes, whose interactions are critical for proper ganglion formation. While the T-cell leukemia homeobox 3 (Tlx3) gene is known to be expressed in placodally-derived sensory neurons and necessary for their differentiation, little was known about Tlx3 expression and/or function in the neural crest-derived component of the developing trigeminal ganglion. By combining lineage labeling with in situ hybridization in the chick embryo, we show that neural crest-derived cells that contribute to the cranial trigeminal ganglion express Tlx3 at a time point that coincides with the onset of ganglion condensation. Importantly, loss of Tlx3 function in vivo diminishes the overall size and abundance of neurons within the trigeminal ganglion. Conversely, ectopic expression of Tlx3 in migrating cranial neural crest results in their premature neuronal differentiation. Taken together, our results demonstrate a critical role for Tlx3 in neural crest-derived cells during chick trigeminal gangliogenesis.

Making Ramón y Cajal proud: Development of cell identity and diversity in the cerebral cortex.

Since the beautiful images of Santiago Ramón y Cajal provided a first glimpse into the immense diversity and complexity of cell types found in the cerebral cortex, neuroscience has been challenged and inspired to understand how these diverse cells are generated and how they interact with each other to orchestrate the development of this remarkable tissue. Some fundamental questions drive the field's quest to understand cortical development: what are the mechanistic principles that govern the emergence of neuronal diversity? How do extrinsic and intrinsic signals integrate with physical forces and activity to shape cell identity? How do the diverse populations of neurons and glia influence each other during development to guarantee proper integration and function? The advent of powerful new technologies to profile and perturb cortical development at unprecedented resolution and across a variety of modalities has offered a new opportunity to integrate past knowledge with brand new data. Here, we review some of this progress using cortical excitatory projection neurons as a system to draw out general principles of cell diversification and the role of cell-cell interactions during cortical development.

Control of successive unequal cell divisions by neural cell fate regulators determines embryonic neuroblast cell size.

Asymmetric cell divisions often generate daughter cells of unequal size in addition to different fates. In some contexts, daughter cell size asymmetry is thought to be a key input to specific binary cell fate decisions. An alternative possibility is that unequal division is a mechanism by which a variety of cells of different sizes are generated during embryonic development. We show here that two unequal cell divisions precede neuroblast formation in the C lineage of Caenorhabditis elegans. The equalisation of these divisions in a pig-1/MELK mutant background has little effect on neuroblast specification. Instead, we demonstrate that let-19/MDT13 is a regulator of the proneural basic helix-loop-helix transcription factor hlh-14/ASCL1 and find that both are required to concomitantly regulate the acquisition of neuroblast identity and neuroblast cell size. Thus, embryonic neuroblast cell size in this lineage is progressively regulated in parallel with identity by key neural cell fate regulators. We propose that key cell fate determinants have a previously unappreciated function in regulating unequal cleavage, and therefore cell size, of the progenitor cells whose daughter cell fates they then go on to specify.

Regulation of early cerebellar development.

The study of cerebellar development has been at the forefront of neuroscience since the pioneering work of Wilhelm His Sr., Santiago Ramón y Cajal and many others since the 19th century. They laid the foundation to identify the circuitry of the cerebellum, already revealing its stereotypic three-layered cortex and discerning several of its neuronal components. Their work was fundamental in the acceptance of the neuron doctrine, which acknowledges the key role of individual neurons in forming the basic units of the nervous system. Increasing evidence shows that the cerebellum performs a variety of homeostatic and higher order neuronal functions beyond the mere control of motor behaviour. Over the last three decades, many studies have revealed the molecular machinery that regulates distinct aspects of cerebellar development, from the establishment of a cerebellar anlage in the posterior brain to the identification of cerebellar neuron diversity at the single cell level. In this review, we focus on summarizing our current knowledge on early cerebellar development with a particular emphasis on the molecular determinants that secure neuron specification and contribute to the diversity of cerebellar neurons.

Transcription factors regulating the specification of brainstem respiratory neurons.

Breathing (or respiration) is an unconscious and complex motor behavior which neuronal drive emerges from the brainstem. In simplistic terms, respiratory motor activity comprises two phases, inspiration (uptake of oxygen, O2) and expiration (release of carbon dioxide, CO2). Breathing is not rigid, but instead highly adaptable to external and internal physiological demands of the organism. The neurons that generate, monitor, and adjust breathing patterns locate to two major brainstem structures, the pons and medulla oblongata. Extensive research over the last three decades has begun to identify the developmental origins of most brainstem neurons that control different aspects of breathing. This research has also elucidated the transcriptional control that secures the specification of brainstem respiratory neurons. In this review, we aim to summarize our current knowledge on the transcriptional regulation that operates during the specification of respiratory neurons, and we will highlight the cell lineages that contribute to the central respiratory circuit. Lastly, we will discuss on genetic disturbances altering transcription factor regulation and their impact in hypoventilation disorders in humans.

Tracing and Manipulating Drosophila Cell Lineages Based on CRISPR: CaSSA and CLADES.

Cell lineage defines the mitotic connection between cells that make up an organism. Mapping these connections in relation to cell identity offers an extraordinary insight into the mechanisms underlying normal and pathological development. The analysis of molecular determinants involved in the acquisition of cell identity requires gaining experimental access to precise parts of cell lineages. Recently, we have developed CaSSA and CLADES, a new technology based on CRISPR that allows targeting and labeling specific lineage branches. Here we discuss how to better exploit this technology for lineage studies in Drosophila, with an emphasis on neuronal specification.

Recessive PRDM13 mutations cause fatal perinatal brainstem dysfunction with cerebellar hypoplasia and disrupt Purkinje cell differentiation.

Pontocerebellar hypoplasias (PCHs) are congenital disorders characterized by hypoplasia or early atrophy of the cerebellum and brainstem, leading to a very limited motor and cognitive development. Although over 20 genes have been shown to be mutated in PCHs, a large proportion of affected individuals remains undiagnosed. We describe four families with children presenting with severe neonatal brainstem dysfunction and pronounced deficits in cognitive and motor development associated with four different bi-allelic mutations in PRDM13, including homozygous truncating variants in the most severely affected individuals. Brain MRI and fetopathological examination revealed a PCH-like phenotype, associated with major hypoplasia of inferior olive nuclei and dysplasia of the dentate nucleus. Notably, histopathological examinations highlighted a sparse and disorganized Purkinje cell layer in the cerebellum. PRDM13 encodes a transcriptional repressor known to be critical for neuronal subtypes specification in the mouse retina and spinal cord but had not been implicated, so far, in hindbrain development. snRNA-seq data mining and in situ hybridization in humans show that PRDM13 is expressed at early stages in the progenitors of the cerebellar ventricular zone, which gives rise to cerebellar GABAergic neurons, including Purkinje cells. We also show that loss of function of prdm13 in zebrafish leads to a reduction in Purkinje cells numbers and a complete absence of the inferior olive nuclei. Altogether our data identified bi-allelic mutations in PRDM13 as causing a olivopontocerebellar hypoplasia syndrome and suggest that early deregulations of the transcriptional control of neuronal fate specification could contribute to a significant number of cases.

Multiple Functions of the Dmrt Genes in the Development of the Central Nervous System.

The Dmrt genes encode the transcription factor containing the DM (doublesex and mab-3) domain, an intertwined zinc finger-like DNA binding module. While Dmrt genes are mainly involved in the sexual development of various species, recent studies have revealed that Dmrt genes, which belong to the DmrtA subfamily, are differentially expressed in the embryonic brain and spinal cord and are essential for the development of the central nervous system. Herein, we summarize recent studies that reveal the multiple functions of the Dmrt genes in various aspects of vertebrate neural development, including brain patterning, neurogenesis, and the specification of neurons.

Transcription Factors That Control Behavior-Lessons From C. elegans.

Behavior encompasses the physical and chemical response to external and internal stimuli. Neurons, each with their own specific molecular identities, act in concert to perceive and relay these stimuli to drive behavior. Generating behavioral responses requires neurons that have the correct morphological, synaptic, and molecular identities. Transcription factors drive the specific gene expression patterns that define these identities, controlling almost every phenomenon in a cell from development to homeostasis. Therefore, transcription factors play an important role in generating and regulating behavior. Here, we describe the transcription factors, the pathways they regulate, and the neurons that drive chemosensation, mechanosensation, thermosensation, osmolarity sensing, complex, and sex-specific behaviors in the animal model Caenorhabditis elegans. We also discuss the current limitations in our knowledge, particularly our minimal understanding of how transcription factors contribute to the adaptive behavioral responses that are necessary for organismal survival.

Defining the nature of human pluripotent stem cell-derived interneurons via single-cell analysis.

The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes.

Single-cell transcriptomics of the early developing mouse cerebral cortex disentangle the spatial and temporal components of neuronal fate acquisition.

In the developing cerebral cortex, how progenitors that seemingly display limited diversity end up producing a vast array of neurons remains a puzzling question. The prevailing model suggests that temporal maturation of progenitors is a key driver in the diversification of the neuronal output. However, temporal constraints are unlikely to account for all diversity, especially in the ventral and lateral pallium where neuronal types significantly differ from their dorsal neocortical counterparts born at the same time. In this study, we implemented single-cell RNAseq to sample the diversity of progenitors and neurons along the dorso-ventral axis of the early developing pallium. We first identified neuronal types, mapped them on the tissue and determined their origin through genetic tracing. We characterised progenitor diversity and disentangled the gene modules underlying temporal versus spatial regulations of neuronal specification. Finally, we reconstructed the developmental trajectories followed by ventral and dorsal pallial neurons to identify lineage-specific gene waves. Our data suggest a model by which discrete neuronal fate acquisition from a continuous gradient of progenitors results from the superimposition of spatial information and temporal maturation.

RUES2 hESCs exhibit MGE-biased neuronal differentiation and muHTT-dependent defective specification hinting at SP1.

RUES2 cell lines represent the first collection of isogenic human embryonic stem cells (hESCs) carrying different pathological CAG lengths in the HTT gene. However, their neuronal differentiation potential has yet to be thoroughly evaluated. Here, we report that RUES2 during ventral telencephalic differentiation is biased towards medial ganglionic eminence (MGE). We also show that HD-RUES2 cells exhibit an altered MGE transcriptional signature in addition to recapitulating known HD phenotypes, with reduced expression of the neurodevelopmental regulators NEUROD1 and BDNF and increased cleavage of synaptically enriched N-cadherin. Finally, we identified the transcription factor SP1 as a common potential detrimental co-partner of muHTT by de novo motif discovery analysis on the LGE, MGE, and cortical genes differentially expressed in HD human pluripotent stem cells in our and additional datasets. Taken together, these observations suggest a broad deleterious effect of muHTT in the early phases of neuronal development that may unfold through its altered interaction with SP1.

Neuronal specification in C. elegans: combining lineage inheritance with intercellular signaling.

The nervous system is composed of a high diversity of neuronal types. How this diversity is generated during development is a key question in neurobiology. Addressing this question is one of the reasons that led Sydney Brenner to develop the nematode C. elegans as a model organism. While there was initially a debate on whether the neuronal specification follows a 'European' model (determined by ancestry) or an 'American' model (determined by intercellular communication), several decades of research have established that the truth lies somewhere in between. Neurons are specified by the combination of transcription factors inherited from the ancestor cells and signaling between neighboring cells (especially Wnt and Notch signaling). This converges to the activation in newly generated postmitotic neurons of a specific set of terminal selector transcription factors that initiate and maintain the differentiation of the neuron. In this review, we also discuss the evolution of these specification mechanisms in other nematodes and beyond.

Muscle-selective RUNX3 dependence of sensorimotor circuit development.

The control of all our motor outputs requires constant monitoring by proprioceptive sensory neurons (PSNs) that convey continuous muscle sensory inputs to the spinal motor network. Yet the molecular programs that control the establishment of this sensorimotor circuit remain largely unknown. The transcription factor RUNX3 is essential for the early steps of PSNs differentiation, making it difficult to study its role during later aspects of PSNs specification. Here, we conditionally inactivate Runx3 in PSNs after peripheral innervation and identify that RUNX3 is necessary for maintenance of cell identity of only a subgroup of PSNs, without discernable cell death. RUNX3 also controls the sensorimotor connection between PSNs and motor neurons at limb level, with muscle-by-muscle variable sensitivities to the loss of Runx3 that correlate with levels of RUNX3 in PSNs. Finally, we find that muscles and neurotrophin 3 signaling are necessary for maintenance of RUNX3 expression in PSNs. Hence, a transcriptional regulator that is crucial for specifying a generic PSN type identity after neurogenesis is later regulated by target muscle-derived signals to contribute to the specialized aspects of the sensorimotor connection selectivity.

Cellular Automata Modeling of Stem-Cell-Driven Development of Tissue in the Nervous System.

Mathematical and computational modeling enables biologists to integrate data from observations and experiments into a theoretical framework. In this review, we describe how developmental processes associated with stem-cell-driven growth of tissue in both the embryonic and adult nervous system can be modeled using cellular automata (CA). A cellular automaton is defined by its discrete nature in time, space, and state. The discrete space is represented by a uniform grid or lattice containing agents that interact with other agents within their local neighborhood. This possibility of local interactions of agents makes the cellular automata approach particularly well suited for studying through modeling how complex patterns at the tissue level emerge from fundamental developmental processes (such as proliferation, migration, differentiation, and death) at the single-cell level. As part of this review, we provide a primer for how to define biologically inspired rules governing these processes so that they can be implemented into a CA model. We then demonstrate the power of the CA approach by presenting simulations (in the form of figures and movies) based on building models of three developmental systems: the formation of the enteric nervous system through invasion by neural crest cells; the growth of normal and tumorous neurospheres induced by proliferation of adult neural stem/progenitor cells; and the neural fate specification through lateral inhibition of embryonic stem cells in the neurogenic region of Drosophila.

Human Neural Stem Cells Flown into Space Proliferate and Generate Young Neurons.

Here we demonstrate that human neural stem cells (NSCs) proliferate while in space and they express specific NSC markers after being in space. NSCs displayed both higher oxygen consumption and glycolysis than ground controls. These cells also kept their ability to become young neurons. Electrophysiological recordings of space NSC-derived neurons showed immature cell membrane properties characterized by small capacitance and very high input resistance. Current injections elicited only an incipient action potential. No spontaneous synaptic events could be detected, suggesting their immature status even though most recorded cells displayed complex morphology and numerous cell processes. Ascertaining the origin of the NSCs' increased energy requirement is of the essence in order to design effective measures to minimize health risks associated with long-duration human spaceflight missions.

Regulation of Neuronal Differentiation, Function, and Plasticity by Alternative Splicing.

Posttranscriptional mechanisms provide powerful means to expand the coding power of genomes. In nervous systems, alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of transcripts. Thus, alternative splicing programs play instructive roles in the development of neuronal cell type-specific properties, neuronal growth, self-recognition, synapse specification, and neuronal network function. Here we discuss the most recent genome-wide efforts on mapping RNA codes and RNA-binding proteins for neuronal alternative splicing regulation. We illustrate how alternative splicing shapes key steps of neuronal development, neuronal maturation, and synaptic properties. Finally, we highlight efforts to dissect the spatiotemporal dynamics of alternative splicing and their potential contribution to neuronal plasticity and the mature nervous system.

Embryology.

With the growing recognition of the extent and prevalence of human cerebellar disorders, an understanding of developmental programs that build the mature cerebellum is necessary. In this chapter we present an overview of the basic epochs and key molecular regulators of the developmental programs of cerebellar development. These include early patterning of the cerebellar territory, the genesis of cerebellar cells from multiple spatially distinct germinal zones, and the extensive migration and coordinated cellular rearrangements that result in the formation of the exquisitely foliated and laminated mature cerebellum. This knowledge base is founded on extensive analysis of animal models, particularly mice, due in large part to the ease of genetic manipulation of this important model organism. Since cerebellar structure and function are largely conserved across species, mouse cerebellar development is highly relevant to humans and has led to important insights into the developmental pathogenesis of human cerebellar disorders. Human fetal cerebellar development remains largely undescribed; however, several human-specific developmental features are known which are relevant to human disease and underline the importance of ongoing human fetal research.

Roles of Retinoic Acid Signaling in Shaping the Neuronal Architecture of the Developing Amphioxus Nervous System.

The morphogen retinoic acid (RA) patterns vertebrate nervous systems and drives neurogenesis, but how these functions evolved remains elusive. Here, we show that RA signaling plays stage- and tissue-specific roles during the formation of neural cell populations with serotonin, dopamine, and GABA neurotransmitter phenotypes in amphioxus, a proxy for the ancestral chordate. Our data suggest that RA signaling restricts the specification of dopamine-containing cells in the ectoderm and of GABA neurons in the neural tube, probably by regulating Hox1 and Hox3 gene expression, respectively. The two Hox genes thus appear to serve distinct functions rather than to participate in a combinatorial Hox code. We were further able to correlate the RA signaling-dependent mispatterning of hindbrain GABA neurons with concomitant motor impairments. Taken together, these data provide new insights into how RA signaling and Hox genes contribute to nervous system as well as to motor control development in amphioxus and hence shed light on the evolution of these functions within vertebrates.

Repression by PRDM13 is critical for generating precision in neuronal identity.

The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development.