The Generation Of Neuronal Cell Diversity

Published Date: 02 Nov 2017

Abstract

The generation of neuronal cell diversity is controlled by interdependent mechanisms, including cell intrinsic programs and environmental cues. During individuals` development, the astonishing variety of neurons is originated according to a precise timetable that is managed by complex networks of genes specifying individual type of neurons. Different neurons express specific sets of transcription factors and they can be recognized by morphological characteristics and spatial localization, but most importantly, they connect to each other and form functional units in a stereotyped fashion. This connectivity depends mostly on selective cell adhesion that is strictly regulated. While intrinsic factors specifying neuronal temporal identity have been extensively studied, extrinsic signaling that acts as the temporal factor to control neuronal temporal identity switch has not been shown. Our data demonstrate that pulses of steroid hormone act as a temporal cue to fine-tuning neuronal cell differentiation. Here we also provide evidence that extrinsic cytokine signaling acts as a spatial code in the process. Particularly, in Drosophila mushroom bodies, neuronal identity transition is controlled by steroid-dependent miRNAs that regulate spatially distributed cytokine-dependent signaling factors that in turn modulate cell adhesion. A new era of neuronal plasticity assessment via managing of external temporal cues such as hormones and cytokines that specify individual types of neurons might open a new possibilities for brain regenerative therapeutics.

Multiplicity of neuron types is generated by intrinsic codes

The development of multiple compartments of the brain is a highly orchestrated process where commitment of certain types of neurons to specific zones, layers and compartments is linked to the developmental stage, at which neurons are generated 1, 2. During the last few years, significant progress has been made in the discovery of genes that identify and control development of different neuronal subtypes (reviewed in 3-5). A subsequent series of intrinsic signaling programs are described in invertebrate and vertebrate organisms where neuronal progenitors in a time-dependent manner progressively acquire specific identity via expression of unique sets of genes that orchestrate the generation of the multiple projection neuron subtypes.

Like in vertebrates, in Drosophila neurons are born from neuronal stem cells that produce different types of neurons depending on embryonic anterior-posterior and dorsal-ventral polarity that establishes gradients of morphogenes and induces expression of gap, pair-rule and Hox genes that subsequently assemble a set of differentially expressed transcription factors 6-11. Following the lineage specification, the neuronal stem cell generates a characteristic set of neuron subtypes 12-14, the exact birthdate of specialized neurons suggests an interaction between temporal cues and neuron-intrinsic cell fate factors. Despite the broad data about existence of these intrinsic programs, it is important to note that the extrinsic temporal determinants of differential morphogenesis have not been revealed in any organism. We discovered that in Drosophila steroid hormones regulate the chronological neuronal identity switch that is executed by steroid-dependent miRNAs (Figure 1).

Steriod hormones regulate an chronological neurogenesis in Drosophila

As a model to study extended neurogenesis we use Drosophila learning center or mushroom body (MB) neurons that are responsible for olfactory learning and memory 15. MB neuron subtypes are generated in the same lineages and specified in a birth-order-dependent fashion 12. MB ? and ?�/?� neurons are produced by type I neuroblasts during larval stages, while ?/? neurons are born after metamorphosis from larval to pupal stages. This MB neuron diversication is coincident to key developmental time periods (Figure 1). In Drosophila there are two key systemic developmental timers, steroid ecdysone and juvenile hormone 16 that synchronize the behavioral, genetic, and morphological changes associated with developmental transitions and the establishment of reproductive maturity 17-25. Pulses of the steroid hormone ecdysone trigger major postembryonic developmental transitions, including molting and metamorphosis 16. Ecdysone interacts with a heterodimer of EcR and USP - two members of nuclear receptor superfamily 26. This complex directly induces expression of primary-response targets, which in turn multiply hormonal signal by regulation of secondary-response gene transcription. These mechanisms specify stage- and tissue- specific responses to each developmentally regulated ecdysone pulse. Moreover, ecdysone signaling is patterned spatially as well as temporally; depending on the cell type and the developmental stage, the ecdysone receptor complex binds different co-activators or co-repressors that can have other binding partners that are regulated by additional signaling pathways. For example, the BTB transcription factor Abrupt attenuates ecdysone signaling by binding to its co-activator Taiman 27 and we showed that this interaction plays an important role in cell non-autonomous regulation of early germline progeny differentiation 25. Furthermore, other signaling pathways (Insulin, TGF-?, JAK/STAT) interact with ecdysone pathway components to further fine-tune the cell-type specific function 27-29. This additional level of combinatorial possibilities allows for a highly managed regulation of gene expression by the systemic signaling. In the brain, it has been shown that ecdysone is responsible for ? neuron remodeling during metamorphosis 30 and we found that ecdysone signaling is also required for ?�/?� to ?/? temporal identity switch that is accomplished via miRNAs to guarantee the specificity of this global endocrine signaling for differentiation of a certain type of neurons in the developing Drosophila brain 31.

Hormones and microRNAs

Development of the living organism is ordered into discrete temporal stages, each is characterized by the unique program of gene expression that controls tissue formation and differentiation. MicroRNAs were first found because of their role in the regulation of developmental staging of the nemathode C. elegans 32, 33. Multiple studies in insects also suggest an important role for miRNAs in the coordination of the developmental transitions; depletion of Dicer-1 (protein required for miRNAs biogenesis) in B. germanica 34 and mutations in Drosophila miRNAs let-7 and miR-125 impair regulation of metamorphic processes 35, 36. The temporal regulation of expression of many other miRNAs is mediated by developmentally controlled hormonal signals. For example, the upregulation of miR-100, miR-125 and let-7 encoded by the miRNA let-7-C locus and downregulation of miR-34 37, miR-14 38 and miR-8 39 in Drosophila require the steroid hormone ecdysone. Recent work from Chawla and Sokol 40 identified and mapped three Ecdysone Response Elements within the let-7-C locus proving that miRNAs can be first-response targets of the hormonal signaling. Importantly, not only hormones regulate miRNA expression; miR-14 has been identified to mediate a positive autoregulatory loop of Ecdysone Receptor that amplifies ecdysone response 38, while miRNA bantam activity in ecdysone producing cells represses hormone production and by that promotes systemic growth 41. Number of studies in vertebrate models and cell cultures as well show relationships between hormones and miRNAs. Glucocorticoids influence a variety of physiological processes in vertebrates, including adaptation to stress, metabolism, immunity and neuronal development. Kawashima et al. 42 show that glucorticosterods regulate levels of brain-derived neurotrophic factor, BDNF via suppression of miR-132 expression, which possibly contributes to the regulation of synaptic plasticity in the brain. On the other hand, miRs -18 and -124a can regulate levels of corticosteroid receptor and therefore modulate downstream effectors of this hormonal signaling 43. Recent work from Huang et al. 44 demonstrates that the miR-21 promoter has a thyroid hormone response element that allows miRNA to be activated in response to hormonal stimuli. Thyroid hormone in vertebrates is an important regulator of development, differentiation and growth. Overactivation of miR-21 promotes hepatoma cell migration and invasion, analogous of that observed with thyroid hormone stimulation. In the breast cancer the estrogen receptor ? (ER?) binds the miR-221-222 transcription start site and recruits co-reppressors to suppress their transcriptional activity 45, while microRNAs miR-191 and miR-425 are upregulated via estrogen-mediated activation 46. Other study shows that miR-221-222 acts as a negative regulator for ER? 47 supporting the idea for the existence of negative regulatory loop involving miRNAs and hormonal receptors.

Together these data show that hormones and miRNAs are prone to work together in regulation of multiple processes. On one hand, cell-specific miRNAs can be used as additional factors that fine-tune the specificity of cellular responses to global hormonal signaling, on the other hand, miRNAs are also involved in feedforward and feedback loops to readjust the precision of this systemic signaling in a given cell type.

?

MicroRNAs in the brain

Biogenesis of miRNAs exhibits specific temporal and spatial profiles in different types of cells and tissues and therefore affects a wide range of biological functions. Conditional knockout of Dicer has been extensively used to address the collective role for miRNAs in specific tissues and cell types in mice. The essential functions for the miRNA pathway have been uncovered in the brain: miRNAs regulate neuronal development and synaptic plasticity, oligodendroglia differentiation and myelin formation, are implicated in brain tumor development and in the regulation of neurodevelopmental and neurodegenerative disorders 48-57. The role of specific miRNAs in the regulation of embryonic and adult neurogenesis, in particular in the proliferation and differentiation of neural stem cells is emerging. Recent work from Parsons et al. 53 provided a genome-scale profiling of miRNA differential expression patterns in human embryonic stem cell neuronal lineage-specific progression. This allowed identifying molecular miRNA signatures for human embryonic neurogenesis: the in vitro neuroectoderm-originated human neuronal cells acquire their identity by down-regulation of pluripotence-associated miRNAs (such as hsa-miR-302 family). In addition, induction of high levels of expression of miRNAs required for regulation of human CNS development (such as hsa-miR-10 and let-7) occurs in a stage-specific manner. In similar studies Stappert et al. 54 demonstrated that time-controlled modulation of specific miRNA activities not only regulates human neural stem cell self-renewal and differentiation but also contributes to the development of defined neuronal subtypes; hence miR-125b and miR-181 promotes and miR-181a* inhibits generation of dopaminergic fate neurons. Boissart et al. 49 found that miR-125 potentiates early neural specification of human embryonic stem cells by regulating SMAD4, a key factor for pluripotent stem cell lineage commitment. Neuron-enriched (miR-376a and miR-434) and glia-enriched (miR-223, miR-146a, miR-19 and miR-32) microRNAs were identified using primary cultures derived from P1 rat cortex 51. MicroRNAs have been also found to direct specific brain regions development during embryogenesis. Nowakowski et al. 52 showed that miR-92b is involved in the regulation of the number of intermediate progenitors populations in mice brain that give rise to the cerebral cortical neurons.

Number of studies in vertebrates reveals the role for miRNAs in the regulation of adult neurogenesis that is largely restricted to two major brain regions: subventricular zone of the lateral ventricle and subventricular zone of the dentate gyrus, in the hippocampus. MicroRNAs let-7b 58, miR-9 56, miR-106b-25 cluster 59, miR-137 60, miR-184 61; miR-124 62 and their specific targets were identified to regulate neural cell proliferation and/or neuronal differentiation during adulthood. Latest studies from Liu et al. 63 uncovered the molecular mechanism by which miR17-92 cluster regulates ischemia-induced neural progenitor cell proliferation which stimulates adult neurogenesis after injury. It has been discovered that stroke substantially upregulates miR17-92 cluster expression in neural progenitor cells of the adult mouse. Overexpression of miR17-92 cluster in the cell culture and in vivo significantly increased cell proliferation, whereas inhibition of individual members of miR17-92 cluster, miR-18a and miR-19a, suppressed cell proliferation and increased cell death. Subventricular zone neuronal fate is determined by miR-124 48: in vivo inhibition of miR-124 causes block in neurogenesis and leads to accumulation of ectopic cells with astrocyte characteristics (neural stem cells) in the olfactory bulb, while upon miR-124 overexpression neural stem cells are not maintained in the subventricular zone of mouse brain and neurogenesis is lost.

Studies from Drosophila 55 revealed that this evolutionary ancient miR-124 controls neural stem cells proliferation by targeting anachronism - an inhibitor of neuroblast proliferation. Drosophila mutant lacking miR-124 shows reduced proliferative activity of neuronal progenitor cells and decreased production of adult postmitotic neurons. We showed that ecdysteroid signaling induces expression of let-7-C in Drosophila brain, which is required for proper differentiation of the last-born neurons in the learning center of the brain. Let-7 deficiency or ecdysone signaling deficit leads to MB morphological defects that result in learning and memory disability 31.

Involvement of miRNAs in regulation of neuronal development, plasticity and maintenance provides a new additional layer of gene regulation, which has an effect on nervous system functions and contributes to neurological diseases. These new findings also propose miRNAs as possible candidates for innovative brain therapies. However, since the general role for miRNAs is the transcriptional repression of their targets, upcoming studies should be focused on finding functional miRNA-target pairs that are also defined at the spatiotemporal level.

BTB transcription factors as temporal codes

We established a spatiotemporal connection between the ecdysteroid-induced miRNA let-7 and its target, the BTB transcription factor Abrupt in the developing brain 31. BTB/POZ zinc finger factors are a class of nuclear DNA-binding proteins containing the BTB domain, which was first identified as a conserved element in the developmentally regulated Drosophila proteins Broad-complex, Tramtrack and Bric-a-brac 64. The BTB is a protein-protein interaction motif found in hundreds of different proteins in organisms ranging from yeast to humans involved in the regulation of gene expression through the local control of chromatin conformation and the recruitment of degradation targets to E3 ubiquitin ligase complexes ligases 65, 66. Interestingly, the BTB domain can form dimers and mediate interactions with non-BTB domain containing proteins and this ability to establish stable and transient interactions. This explains the ability of BTB containing proteins to participate in multiple processes and implies that management of their proper levels is of a particular significance 66.

BTB/POZ domain zinc finger factors were linked to broad developmental processes in vertebrates and invertebrates: chromatin remodeling, cancer development and intriguingly, regulation of cell fate specification in the nervous system 64-70. For example, the BTB/POZ zinc-finger transcription factor-encoding gene Rp58 is required for the correct differentiation of neural progenitors into neurons, since its neural-specific deletion results in severe cerebellar hypoplasia and developmental failure of several neuronal types 69. By coherently repressing multiple proneurogenic genes in a timely manner this BTB protein supports neuronal differentiation and brain growth 70. During embryonic development of the murine cerebral cortex another mammalian BTB factor, HOF is specifically expressed in immature non-dividing cells and is down-regulated in differentiated cells of the hippocampus; importantly, it is one of the factors that might be involved in early definition of hippocampal compartment within the neocortex 68.

Similarly, in the Drosophila nervous system several BTB/POZ domain zinc finger transcription factors have been implicated in specifying neuronal and glial cell lineages. For example, Tramtrack proteins transcriptionally repress genes that promote transformation of neuronal support cells into neurons 71, 72, while Lola, Fruitless, Abrupt, and Chinmo are intrinsically required for development of different subsets of neurons 31, 67, 73-78. Such data provide evidence that BTB/POZ zinc-finger proteins play an important role in a transcriptional program that controls differentiation of progenitors into neurons. Since the growth and organization of the brain is tightly correlated with the speed of the whole organisms development, it implies that neuron differentiation should be responsive to external temporal cues. Interestingly, the neuronal temporal identity of Drosophila mushroom body neurons is governed by two BTB transcription factors, Chinmo and Abrupt and both of them are subjects to miRNA-mediated regulation 31, 77, 78. We found that this regulation is chronologically induced by systemic steroid signaling that controls the major larva-to-pupa transition during Drosophila development, which also coincides with the time-point when the last-born neurons are generated 31. This demonstrated for the first times that differential neurogenesis is hierarchically regulated by extrinsic systemic signaling, which, in chronological manner, adjusts programs of intrinsic temporal determinants of neuronal cell fate and that BTB transcripton factors play a role as temporal codes in the process..

Next, we wanted to understand whether intercellular environmental signaling, such as extrinsic cell-cell signaling would also cooperate to fine-tune the outcome of differential neurogenesis.

Concerted action of cytokines and steroids in differential neurogenesis

JAK/STAT signaling is involved in Abrupt regulation during MB development

Interestingly, let-7 target Abrupt that is expressed in MBs is associated with the evolutionary conserved JAK/STAT signaling pathway that plays key roles in multiple developmental and physiological processes in the brain, ranging from the regulation of neurogenesis and stem cell fate to memory formation 79-81. In the adult brain, endogenous cytokine levels are very low under normal physiological conditions, however various types of injuries, including trauma, seizures, and ischemia induce an increase of cytokine ligand levels, which in turn promotes neuronal stem cell self-renewal (Bauer and Patterson, 2006). For example, during development, some neuroepithelial cells become neuroblasts and generate the neuronal and glial cells, and in the Drosophila optic lobe the timing of this transition is negatively regulated by JAK/STAT signaling. Secretion of the JAK/STAT ligand Unpaired (Upd) shapes an activity gradient in the neuroepithelium and negatively regulates the progression of the proneural wave 82. JAK/STAT signaling is further integrated with the Notch and EGFR signals to balance neuroblast self-renewal and neuron differentiation 80, 82. Transcription factor Ab has been shown previously to be negatively regulated by the JAK/STAT signaling pathway in ovaries 27, 83. Following this idea we evaluated whether JAK/STAT plays a role in Ab regulation during MB development. We used a 10xSTAT-GFP reporter line (Fig. 2A, C) and antibodies against STAT92E (Fig. 2D) to visualize JAK/STAT signaling activity in the developing brain. At the larval stage we observed GFP signal predominantly in neuroblasts (Miranda positive cells) and glial cells (Repo positive, Fig.2A). Since at the pupal and pharate stage brains only four (from each side) mushroom body neuroblasts continue to divide 84, GFP signal indicating JAK/STAT activity was restricted to these four NBs and glial cells ( Fig. 2C); similar pattern was detected with STAT-92E antibodies (Fig. 2D). Based on previously described expression patterns for Ab and miRNA let-7 31 that was restricted to ?, ?�/?� and ?/? MB lobe neurons respectively, and currently observed JAK/STAT signaling activity in the MB neuroblasts, we hypothesized the possibility of Ab regulation by the spatially distributed cytokine signaling that takes place in the MB neural stem cells (Fig. 2B). Downregulation of JAK/STAT signaling via expression of dominant negative form of dome specifically in the neuroblasts resulted in changed Ab expression pattern in the MB cell body clasters and in appearance of ectopic Ab protein in some of the neuroblasts (Fig. 2E,F). During Drosophila development MBNs are continuously dividing and give rise to mushroom body neurons (Kenyon cells) that are clustered into three types of mushroom body lobes (?, ?�/?� and ?/?) with distinct axonal projection patterns. We used FasII antibodies as a molecular marker for ? and ?/? MB axons to evaluate whether dowregulation of JAK/STAT signaling or overactivation of the transcription factor Abrupt in the MBNs affects overall MB morphology. We observed that overexpression of a dominant negative version of dome as well as UAS-STAT RNAi using neuroblast-specific driver lines (inscGal4 and worGal4) indeed caused morphological changes detected in the adult mushroom bodies stained with anti-FasII; MBs with slim ?/? lobes and fused ?-lobes (Fig. 2G-H,J-K; Table 1) were observed. Similar MB morphological defects were identified upon overexpression of Ab in the neuroblasts (Fig. 2I-J,L; Table 1). This evidence supports the hypothesis that spatially distributed JAK/STAT signaling acts as a repressor of the transcription factor Ab in neuronal stem cells and this downregulation is critical for proper neurogenesis.

The specificity of JAK/STAT cytokine signaling action depends on the downstream-activated effector genes, for example, the BTB-zinc finger protein Chinmo regulates stem cell self-renewal 85 and governs neuroblast temporal identity 78. Intriguingly, Chinmo confers all of the transitions in MB temporal identity, except for ?/? neurons 78, that, as we show here, is coordinated by the ecdysone peak, let-7 expression and Abrupt. Previous data show that the transcription factor Ab acts as a transdetermination factor that, when misexpressed, is sufficient to potentiate even homeotic arista-leg transformation 86. This implies that regulation of Ab expression should be under strict developmental control to guarantee faithful cell fate determination. Our data provide evidence that in the developing brain to ensure proper MB development the temporally induced by ecdysone pulse miRNA let-7 negatively regulates Ab, which potentially can be additionally targeted by the local JAK/STAT cytokine pathway.

Taken together, previous data and our findings demonstrate that neurogenesis is regulated not only by intrinsic factors expressed in progenitors, but also extrinsic cues from neighboring cells and developmentally regulated temporal signals. The interaction between more general developmental and local tissue-specific signaling results in establishment of a robust spatio-temporal pattern of the cell identity specification.

Cell adhesion as a final outcome of differential neuron differentiation

The complexity of the brain is generated by multiple types of neurons that connect to each other in a specialized manner, which often depends on selective cell adhesion 87. Neurons expressing similar cell adhesion proteins not only cluster together to organize brain compartments that have distinct functions, selective cell adhesion is also used for establishing of synaptic connections that allow neurons to communicate and transfer information. Significant alterations in the brain structure and functions are generated even by moderate changes in the quantities of adhesion molecules on the neuronal cell surfaces. Therefore differential cell adhesion is the final aftermath of differential neurogenesis, suggesting that timing and levels of cell adhesion protein expression must be precisely regulated.

Among the most important cell adhesion molecules (CAMs) involved in the development of the nervous system, synaptic plasticity, and cognition and memory are neural cell adhesion molecules (NCAMs) that belong to the immunoglobulin superfamily. Previous data show that levels of human NCAM2 that is primarily expressed in the brain to stimulate neurite outgrowth and facilitate dendritic and axonal compartmentalization are essential for normal brain development 88. For example, the increased expression of NCAM2 as a result of trisomy 21 may cause dosage-related detrimental effects in Down syndrome; also, in genome-wide association studies, NCAM2 was suggested as a candidate gene for the development of autism and Alzheimer disease 89-91 and multiple NCAM1 proteins are differentially altered in bipolar disorder and schizophrenia 92. Besides, NCAMs are known to play a critical role in plasticity of the nervous system and in mechanisms controlling learning and memory and their expression levels are known to be highly susceptible to modulation by stress 93. Moreover, NCAM is involved in some of the bidirectional effects of stress on memory processes, where its increased synaptic expression is facilitating stress actions while its decreased expression is impairing effects of stress on memory consolidation 94.

These data i that regulation of NCAM expression is a prerequisite for proper brain development and function. What genetic machinery regulates precise expression of adhesion molecules in the brain? Ample sets of regulatory elements are required for spatiotemporally restricted expression pattern of a given gene. It is not well-defined what set of transcriptional factors regulates differential expression of different cell adhesion proteins that modulate the degrees to which various neurons adhere to each other and make synapses. In the Drosophila learning center the orthologue of NCAMs, Fasciclin 2 (Fas2) displays specific temporal patterns of expression that plays a significant role in the spatial segregation of MB neurons. Low levels of Fas2 is detected in the ? and high levels in the ?/?, but not ?�/?� lobes 95-97. Fas2 provides specific adhesive codes among MB neurons preventing them from intermingling and assuring distinct MB lobes formation. We showed that the transcription factor Abrupt suppresses Fas2 expression in the earlier-born ?�/?� neurons, while steroid-induced miRNA via downregulation of Abrupt allows this critical adhesion molecule to be expressed in the late-born ?/? neurons. Thus, the precise Fas2 expression is essential for proper MB morphology and function 31.

Together these data show that NCAMs are multifunction proteins involved in neurodevelopment and neurogenesis and their expression levels are critical dendritic and axonal compartmentalization and synaptic plasticity. This puts differential cell adhesion as a fundamental mechanism of neuronal cell differentiation that controls the finest aspects of neuronal specification. Once a specific neuron is born, it must recognize and join other neurons of the appropriate type to assemble into a specific brain compartment that normally is determined and maintained by the system of preferential cell affinities. Even more, neurons send out axons and dendrites that via differential cell adhesion make synapses with other neurons. However, neurons do not simply remain passively stuck together; instead, the new synapses are generated and actively maintained by selective adhesions that neurons create and gradually adjust, which contributes to plasticity of the nervous system. We show that miRNAs are mediators between extrinsic temporal cues and intrinsic spatiotemporal codes that determine the precision of neuronal adhesiveness during brain development, it would be important to explore the role of these factors in the adult brain plasticity.

Interestingly, it has been proposed that the increased stickiness of human neurons might explain the accelerated evolution of the human brain beyond the brains of primates98. Another factor that distinguishes humans from other primates is that developmental profiles of miRNAs, as well as their target genes, show the fastest rates of human-specific evolutionary change, which allows for the faster evolutionary rate in divergence of developmental patterns 99. One of the key features of the miRNA function is that miRNAs normally do not turn on-off their target genes, but just modulate their expression. This allows building novel networks between newly originated genes and miRNAs softly, without killing the organism. Analysis of recently originated brain genes in Drosophila showed that numerous newly evolved genes are expressed in the brain and all of the MB-positive new genes are expressed in the ?/?, but not in more ancestral ? and ?�/?� lobes 100. Since miRNAs and newly evolved genes are frequently co-expressed in the brain, the hypothesis can be put forward that the establishment of novel sets of spatiotemporal codes for differential neurogenesis that are gently fine-tuned by miRNAs is a common mechanism that might contribute to the phenotypic evolution of behavior and individual plasticity of the nervous system. Management of programs of genes that temporally specify individual subtypes of neurons could help to evaluate the true limits of progenitor plasticity within the developing and adult brain and initiate a new phase of plasticity assessment.