Diverse Neuronal Subtypes Research Articles

Functional Anatomy of the Sensory Nervous System: Updates From the Neuroscience Bench.

The simple tripartite classification of sensory neurons as A-beta, A-delta, and C fibers fails to convey the complexity of the neurons that encode stimuli as diverse as the texture of a surface, the location of a pinprick, or the direction of hair movement as a breeze moves across the skin. It has also proven to be inadequate when investigating the molecular mechanisms underlying pain, which can encompass any combination of chemical, tactile, and thermal modalities. Beginning with a brief overview of visceral and sensory neuroanatomy, this review expands upon sensory innervation of the skin as a prime example of the heterogeneity and complexity of the somatosensory nervous system. Neuroscientists have characterized defining features of over 15 subtypes of sensory neurons that innervate the skin of the mouse. This has enabled the study of cell-specific mechanisms of pain, which suggests that diverse sensory neuron subtypes may have distinct susceptibilities to toxic injury and different roles in pathologic mechanisms underlying altered sensation. Leveraging this growing body of knowledge for preclinical trials and models of neurotoxicity can vastly improve our understanding of peripheral nervous system dysfunction, advancing the fields of toxicologic pathology and neuropathology alike.

PDK1 Regulates Transition Period of Apical Progenitors to Basal Progenitors by Controlling Asymmetric Cell Division.

The six-layered neocortex consists of diverse neuron subtypes. Deeper-layer neurons originate from apical progenitors (APs), while upper-layer neurons are mainly produced by basal progenitors (BPs), which are derivatives of APs. As development proceeds, an AP generates two daughter cells that comprise an AP and a deeper-layer neuron or a BP. How the transition of APs to BPs is spatiotemporally regulated is a fundamental question. Here, we report that conditional deletion of phoshpoinositide-dependent protein kinase 1 (PDK1) in mouse developing cortex achieved by crossing Emx1Cre line with Pdk1fl/fl leads to a delayed transition of APs to BPs and subsequently causes an increased output of deeper-layer neurons. We demonstrate that PDK1 is involved in the modulation of the aPKC-Par3 complex and further regulates the asymmetric cell division (ACD). We also find Hes1, a downstream effecter of Notch signal pathway is obviously upregulated. Knockdown of Hes1 or treatment with Notch signal inhibitor DAPT recovers the ACD defect in the Pdk1 cKO. Thus, we have identified a novel function of PDK1 in controlling the transition of APs to BPs.

Translating from cancer to the brain: regulation of protein synthesis by eIF4F.

Formation of eukaryotic initiation factor 4F (eIF4F) is widely considered to be the rate-limiting step in cap-dependent translation initiation. Components of eIF4F are often up-regulated in various cancers, and much work has been done to elucidate the role of each of the translation initiation factors in cancer cell growth and survival. In fact, many of the basic mechanisms describing how eIF4F is assembled and how it functions to regulate translation initiation were first investigated in cancer cell lines. These same eIF4F translational control pathways also are relevant for neuronal signaling that underlies long-lasting synaptic plasticity and memory, and in neurological diseases where eIF4F and its upstream regulators are dysregulated. Although eIF4F is important in cancer and for brain function, there is not always a clear path to use the results of studies performed in cancer models to inform one of the roles that the same translation factors have in neuronal signaling. Issues arise when extrapolating from cell lines to tissue, and differences are likely to exist in how eIF4F and its upstream regulatory pathways are expressed in the diverse neuronal subtypes found in the brain. This review focuses on summarizing the role of eIF4F and its accessory proteins in cancer, and how this information has been utilized to investigate neuronal signaling, synaptic function, and animal behavior. Certain aspects of eIF4F regulation are consistent across cancer and neuroscience, whereas some results are more complicated to interpret, likely due to differences in the complexity of the brain, its billions of neurons and synapses, and its diverse cell types.

Conversion of Astrocytes and Fibroblasts into Functional Noradrenergic Neurons

Dysfunction of noradrenergic (NA) neurons is associated with a number of neuronal disorders. Diverse neuronal subtypes can be generated by direct reprogramming. However, it is still unknown how to convert non-neuronal cells into NA neurons. Here, we show that seven transcription factors (TFs) (Ascl1, Phox2b, AP-2α, Gata3, Hand2, Nurr1, and Phox2a) are able to convert astrocytes and fibroblasts into induced NA (iNA) neurons. These iNA neurons express the genes required for the biosynthesis, release, and re-uptake of noradrenaline. Moreover, iNA neurons fire action potentials, receive synaptic inputs, and control the beating rate of co-cultured ventricular myocytes. Furthermore, iNA neurons survive and integrate into neural circuits after transplantation. Last, human fibroblasts can be converted into functional iNA neurons as well. Together, iNA neurons are generated by direct reprogramming, and they could be potentially useful for disease modeling and cell-based therapies.

Conditional Synaptic Vesicle Markers for Drosophila.

The release of neurotransmitters from synaptic vesicles (SVs) at pre-synaptic release sites is the principle means by which information transfer between neurons occurs. Knowledge of the location of SVs within a neuron can thus provide valuable clues about the location of neurotransmitter release within a neuron and the downstream neurons to which a given neuron is connected, important information for understanding how neural circuits generate behavior. Here the development and characterization of four conditional tagged SV markers for Drosophila melanogaster is presented. This characterization includes evaluation of conditionality, specificity for SV localization, and sensitivity of detection in diverse neuron subtypes. These four SV markers are genome-edited variants of the synaptic vesicle-specific protein Rab3. They depend on either the B2 or FLP recombinases for conditionality, and incorporate GFP or mCherry fluorescent proteins, or FLAG or HA epitope tags, for detection.

Pivotal roles of Fezf2 in differentiation of cone OFF bipolar cells and functional maturation of cone ON bipolar cells in retina

During development of the retina, common retinal progenitor cells give rise to six classes of neurons that subsequently further diversify into more than 55 subtypes of neuronal subtypes. Here, we have investigated the expression and function of Fezf2, Fez zinc finger family of protein, in the developing mouse retina. Expression of Fezf2 transcripts was strongly observed in the embryonic retinal progenitors at E14.5 and declined quickly in subsequent development of retina. Then, in postnatal stage at around day 8, Fezf2 was transiently expressed then declined again. Loss-of-function analysis using retinas from mice in which Fezf2 coding region was substituted with β-galactosidase showed that Fezf2 is expressed in a subset of cone OFF bipolar cells and required for their differentiation. Using electroretinogram, we found that Fezf2 knockout retina exhibited significantly reduced photopic b-wave, suggesting functional abnormality of cone ON bipolar cells. Furthermore, reduced expression of synaptic protein Trpm1 and structural alteration of ON bipolar cell invagination, both of which affected cone photoreceptor terminal synaptic activity, was identified by transmission electron microscopy and immunohistochemistry, respectively. Taken together, our results show that Fezf2 is indispensable in differentiation of bipolar precursors into cone OFF bipolar cells and in functional maturation of cone ON bipolar cells during development of mouse retina. These results contribute to our understanding of how diversity of neuronal subtypes and hence specificity of neuronal connections are established in the retina by intrinsic cues.

Single-injection ex ovo transplantation method for broad spinal cord engraftment of human pluripotent stem cell-derived motor neurons

BackgroundTransplantation of human pluripotent stem cell (hPSC)-derived neurons into chick embryos is an established preliminary assay to evaluate engraftment potential. Yet, with recent advances in deriving diverse human neuronal subtypes, optimizing and standardizing such transplantation methodology for specific subtypes at their correlated anatomical sites is still required. New methodWe determined the optimal stage of hPSC-derived motor neuron (hMN) differentiation for ex ovo transplantation, and developed a single injection protocol that implants hMNs throughout the spinal cord enabling broad regional engraftment possibilities. ResultsA single injection into the neural tube lumen yielded a 100% chick embryo survival and successful transplantation rate with MN engraftment observed from the rostral cervical through caudal lumbar spinal cord. Transplantation of HB9+/ChAT− hMN precursors yielded the greatest amount of engraftment compared to Pax6+/Nkx6.1+/Olig2+ progenitors or mature HB9+/ChAT+ hMNs. Comparison with existing method(s)Our single injection hMN transplant method is the first to standardize the optimal hMN phenotype for chick embryo transplantation, provide a rubric for engraftment quantification, and enable broad engraftment throughout the spinal cord with a single surgical intervention. ConclusionTransplantation of HB9+/ChAT− hMN precursors into chick embryos of Hamburger Hamilton (HH) stages 15–18 using a single luminal injection confers a high probability of embryo survival and cell engraftment in diverse regions throughout the spinal cord.

Sonic hedgehog in vertebrate neural tube development.

The formation and wiring of the vertebrate nervous system involves the spatially and temporally ordered production of diverse neuronal and glial subtypes that are molecularly and functionally distinct. The chick embryo has been the experimental model of choice for many of the studies that have led to our current understanding of this process, and has presaged and informed a wide range of complementary genetic studies, in particular in the mouse. The versatility and tractability of chick embryos means that it remains an important model system for many investigators in the field. Here we will focus on the role of Sonic hedgehog (Shh) signaling in coordinating the diversification, patterning, growth and differentiation of the vertebrate nervous system. We highlight how studies in chick led to the identification of the role Shh plays in the developing neural tube and how subsequent work, including studies in the chick and the mouse revealed details of the cell intrinsic programs controlling cell fate determination. We compare these mechanisms at different rostral-caudal positions along the neuraxis and discuss the particular experimental attributes of the chick that facilitated this work.

Classifying <i>Drosophila</i> Olfactory Projection Neuron Subtypes by Singlecell RNA Sequencing

How a neuronal cell type is defined and how this relates to its transcriptome are still open questions. The Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: Different PN classes target dendrites to distinct olfactory glomeruli and PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA-sequencing, we comprehensively characterized the transcriptomes of 40 PN classes and unequivocally identified transcriptomes for 6 classes. We found a new lineage-specific transcription factor that instructs PN dendrite targeting. Transcriptomes of closely related PN classes exhibit the largest difference during circuit assembly, but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Genes encoding transcription factors and cell-surface molecules are the most differentially expressed, indicating their central roles in specifying neuronal identity. Finally, we show that PNs use highly redundant combinatorial molecular codes to distinguish subtypes, enabling robust specification of cell identity and circuit assembly.

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing

The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes ofclosely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.

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.

Septal contributions to olfactory bulb interneuron diversity in the embryonic mouse telencephalon: role of the homeobox gene Gsx2

BackgroundOlfactory bulb (OB) interneurons are known to represent diverse neuronal subtypes, which are thought to originate from a number of telencephalic regions including the embryonic dorsal lateral ganglionic eminence (dLGE) and septum. These cells migrate rostrally toward the OB, where they then radially migrate to populate different OB layers including the granule cell layer (GCL) and the outer glomerular layer (GL). Although previous studies have attempted to investigate regional contributions to OB interneuron diversity, few genetic tools have been used to address this question at embryonic time points when the earliest populations are specified.MethodsIn this study, we utilized Zic3-lacZ and Gsx2e-CIE transgenic mice as genetic fate-mapping tools to study OB interneuron contributions derived from septum and LGE, respectively. Moreover, to address the regional (i.e. septal) requirements of the homeobox gene Gsx2 for OB interneuron diversity, we conditionally inactivated Gsx2 in the septum, leaving it largely intact in the dLGE, by recombining the Gsx2 floxed allele using Olig2Cre/+ mice.ResultsOur fate mapping studies demonstrated that the dLGE and septum gave rise to OB interneuron subtypes differently. Notably, the embryonic septum was found to give rise largely to the calretinin+ (CR+) GL subtype, while the dLGE was more diverse, generating all major GL subpopulations as well as many GCL interneurons. Moreover, Gsx2 conditional mutants (cKOs), with septum but not dLGE recombination, showed impaired generation of CR+ interneurons within the OB GL. These Gsx2 cKOs exhibited reduced proliferation within the septal subventricular zone (SVZ), which correlated well with the reduced number of CR+ interneurons observed.ConclusionsOur findings indicate that the septum and LGE contribute differently to OB interneuron diversity. While the dLGE provides a wide range of OB interneuron subtypes, the septum is more restricted in its contribution to the CR+ subtype. Gsx2 is required in septal progenitors for the correct expansion of SVZ progenitors specified toward the CR+ subtype. Finally, the septum has been suggested to be the exclusive source of CR+ interneurons in postnatal studies. Our results here demonstrate that dLGE progenitors in the embryo also contribute to this OB neuronal subtype.

Human astrocytes are distinct contributors to the complexity of synaptic function.

Cellular components of synaptic circuits have been adjusted for increased human brain size, neural cell density, energy consumption and developmental duration. How does the human brain make these accommodations? There is evidence that astrocytes are one of the most divergent neural cell types in primate brain evolution and it is now becoming clear that they have critical roles in controlling synaptic development, function and plasticity. Yet, we still do not know how the precise developmental appearance of these cells and subsequent astrocyte-derived signals modulate diverse neuronal circuit subtypes. Here, we discuss what is currently known about the influence of glial factors on synaptic maturation and focus on unique features of human astrocytes including their potential roles in regenerative and translational medicine. Human astrocyte distinctiveness may be a major contributor to high level neuronal processing of the human brain and act in novel ways during various neuropathies ranging from autism spectrum disorders, viral infection, injury and neurodegenerative conditions.

Sip1 regulates the generation of the inner nuclear layer retinal cell lineages in mammals.

The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.

Distribution of Non-pyramidal Neurons in the Frontal Lobe of Indian Gray Mongoose (Herpestes edwardsii)

Nissl and Golgi staining techniques have been employed to explore the neuronal subtypes in frontal lobe of Indian gray mongoose. A wide study of sixth layer in cerebral cortex of gyrencephalic and lissencephalic brains of mammals pointed out that pyramidal neurons are the dominant projection neuron in the cortical region and contribute to major neuronal circuitry. Frontal lobe well connected with other cortical and subcortical brain structures have been related with coordination of motor behaviour. Studies related to mammalian cortices focused mainly on pyramidal neurons, however, the details for other neuronal classes have gained less attention. Hence the aim of the present study was to explore different types of neuronal classes within frontal lobe of mongoose. Eleven types of neurons were identified in the mongoose frontal isocortex on the basis of morphology as seen in the Golgi-impregnated sections. Diversity of neuronal subtypes in frontal lobe of mongoose adds to the integration ability of sensory inputs and better processing. The specialized neuronal features observed in frontal lobe have been correlated with better decision making and attention skills thereby providing improved skills for prey and predator mode of action in mongoose.

Physiological, pathological, and engineered cell identity reprogramming in the central nervous system.

Multipotent neural stem cells persist in restricted regions of the adult mammalian central nervous system. These proliferative cells differentiate into diverse neuron subtypes to maintain neural homeostasis. This endogenous process can be reprogrammed as a compensatory response to physiological cues, traumatic injury, and neurodegeneration. In addition to innate neurogenesis, recent research has demonstrated that new neurons can be engineered via cell identity reprogramming in non-neurogenic regions of the adult central nervous system. A comprehensive understanding of these reprogramming mechanisms will be essential to the development of therapeutic neural regeneration strategies that aim to improve functional recovery after injury and neurodegeneration. WIREs Dev Biol 2016, 5:499-517. doi: 10.1002/wdev.234 For further resources related to this article, please visit the WIREs website.

Sox2 goes beyond stem cell biology

Diversity in neuronal types provides us with the ability to form complex thoughts and functions. The molecular mechanisms used to generate neuronal subtype diversity in the nervous system are still...

Generation of Regionally Specific Neural Progenitor Cells (NPCs) and Neurons from Human Pluripotent Stem Cells (hPSCs).

Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population capable of long-term expansion and differentiation into a variety of neuronal subtypes. As such, NPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine. Current methods for the generation of NPCs results in cell populations homogenous for pan-neural markers such as SOX1 and SOX2 but heterogeneous with respect to regional identity. In order to use NPCs and their neuronal derivatives to investigate mechanisms of neurological disorders and develop more physiologically relevant disease models, methods for generation of regionally specific NPCs and neurons are needed. Here, we describe a protocol in which exogenous manipulation of WNT signaling, through either activation or inhibition, during neural differentiation of hPSCs, promotes the formation of regionally homogenous NPCs and neuronal cultures. In addition, we provide methods to monitor and characterize the efficiency of hPSC differentiation to these regionally specific cell identities.

Single-base pair differences in a shared motif determine differential Rhodopsin expression.

The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, we compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11-base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli.

Controlling the Regional Identity of hPSC-Derived Neurons to Uncover Neuronal Subtype Specificity of Neurological Disease Phenotypes.

SummaryThe CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs.