Neuronal Cell Fate Research Articles

The Phylogenetic Functional Conservation of  <i>Drosophila</i> Seven-in-Absentia (SINA) E3 Ligase and Its Two Human Paralogs, SIAH1 and SIAH2, in  <i>Drosophila</i> Eye Development

Seven-IN-Absentia (SINA) is the most downstream signaling gatekeeper identified in the Drosophila RAS/EGFR signaling pathway. Underscoring the central importance of Drosophila SINA and human SINA homologs (SIAHs) is its phylogenetic conservation across metazoans. SIAH is a major tumor vulnerability in human cancer. To further delineate the SINA function in Drosophila, a genetic modifier screen was conducted, and 28 new sina mutant alleles were isolated. By sequencing analyses, we identified three invariable amino acid residues in SINA’s RING-domain whose single point mutation ablates SINA function . To demonstrate the functional conservation of this medically important family of RING domain E3 ligases, we compared expression of either wild type (WT) or proteolysis-deficient (PD) SINA/SIAH inhibitors of Drosophila SINAWT/PD and human SIAH1WT/PD /2WT/PD under tissue-specific GAL4-drivers in Drosophila. Our results showed that Drosophila SINA and human SIAH1/2 are functionally conserved. The SINAPD/SIAHPD inhibitors are effective in blocking the RAS-dependent neuronal cell fate determination in Drosophila.

In Vitro Direct Reprogramming of Mouse and Human Astrocytes to Induced Neurons.

Direct neuronal reprogramming, rewiring the epigenetic and transcriptional network of a differentiated cell type to neuron, apart from being a very promising approach for the treatment of brain injury and neurodegeneration, also offers a prime opportunity to investigate the molecular underpinnings of neuronal cell fate determination, as the precise molecular mechanisms that establish neuronal fate and diversity at the transcriptional and epigenetic level are incompletely understood. Recent studies from a number of groups, including ours, have shown that astrocytes can be directly reprogrammed into functional neurons in vitro and in vivo following ectopic overexpression of combinations of transcription factors, neurogenic proteins, miRNAs, and small chemical molecules.In this chapter we describe the protocols for in vitro converting primary cortical astrocytes of mouse and human origin to induced neurons, through forced expression of two neurogenic molecules, either each one alone or in combination: the master regulatory bHLH proneural transcription factor NEUROGENIN-2 (NEUROG2) and the neurogenic protein CEND1. Forced expression of each one of the two neurogenic proteins in primary astrocytes via retroviral gene transfer results in their direct conversion to subtype-specific induced neurons, while simultaneous coexpression of both molecules drives them predominantly toward acquisition of a neural precursor cell (NPC) state. Although mouse and human astrocytes exhibit differences in their reprogramming rate and particular characteristics, they can both get efficiently in vitro transdifferentiated to NPCs and induced neurons upon NEUROG2 or/and CEND1 forced expression using the reprogramming protocols described in the chapter, presenting valuable cellular platforms for mechanistic studies and in vivo applications to restore neurodegeneration.

Immunomodulatory role of the extracellular matrix protein tenascin-C in neuroinflammation.

The extracellular matrix (ECM) consists of a dynamic network of various macromolecules that are synthesized and released by surrounding cells into the intercellular space. Glycoproteins, proteoglycans and fibrillar proteins are main components of the ECM. In addition to general functions such as structure and stability, the ECM controls several cellular signaling pathways. In this context, ECM molecules have a profound influence on intracellular signaling as receptor-, adhesion- and adaptor-proteins. Due to its various functions, the ECM is essential in the healthy organism, but also under pathological conditions. ECM constituents are part of the glial scar, which is formed in several neurodegenerative diseases that are accompanied by the activation and infiltration of glia as well as immune cells. Remodeling of the ECM modulates the release of pro- and anti-inflammatory cytokines affecting the fate of immune, glial and neuronal cells. Tenascin-C is an ECM glycoprotein that is expressed during embryonic central nervous system (CNS) development. In adults it is present at lower levels but reappears under pathological conditions such as in brain tumors, following injury and in neurodegenerative disorders and is highly associated with glial reactivity as well as scar formation. As a key modulator of the immune response during neurodegeneration in the CNS, tenascin-C is highlighted in this mini-review.

Benzothiazole-Based LRRK2 Inhibitors as Wnt Enhancers and Promoters of Oligodendrocytic Fate.

Leucine rich repeat kinase 2 (LRRK2) is an enigmatic enzyme and a relevant target for Parkinson's disease (PD). However, despite the significant amount of research done in the past decade, the precise function of LRRK2 remains largely unknown. Moreover, the therapeutic potential of its inhibitors is in its infancy with the first clinical trial having just started. In the present work, the molecular mechanism of LRRK2 in the control of neurogenesis or gliogenesis was investigated. We designed and synthesized novel benzothiazole-based LRRK2 inhibitors and showed that they can modulate the Wnt/β-catenin signaling pathway. Furthermore, compounds 5 and 14 were able to promote neural progenitors proliferation and drive their differentiation toward neuronal and oligodendrocytic cell fates. These results suggest potential new avenues for the application of LRRK2 inhibitors in demyelinating diseases in which oligodendrocyte cell-death is one of the pathological features.

A universal transportin protein drives stochastic choice of olfactory neurons via specific nuclear import of a sox-2-activating factor

Stochastic neuronal cell fate choice involving notch-independent mechanisms is a poorly understood biological process. The Caenorhabditis elegans AWC olfactory neuron pair asymmetrically differentiates into the default AWCOFF and induced AWCON subtypes in a stochastic manner. Stochastic choice of the AWCON subtype is established using gap junctions and SLO BK potassium channels to repress a calcium-activated protein kinase pathway. However, it is unknown how the potassium channel-repressed calcium signaling is translated into the induction of the AWCON subtype. Here, we identify a detailed working mechanism of how the homeodomain-like transcription factor NSY-7, previously described as a repressor in the maintenance of AWC asymmetry, couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype through the identification of a unique imb-2 (transportin 1) allele. imb-2 loss-of-function mutants are not viable; however, we identify a viable imb-2 allele from an unbiased forward genetic screen that reveals a specific role of imb-2 in AWC olfactory neuron asymmetry. IMB-2 specifically drives nuclear import of NSY-7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box transcription factor SOX-2 for the specification of the AWCON subtype. This study provides mechanistic insight into how NSY-7 couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype. Our findings also provide structure-function insight into a conserved amino acid residue of transportins in brain development and suggest its dysfunction may lead to human neurological disorders.

IRE1 promotes neurodegeneration through autophagy-dependent neuron death in the Drosophila model of Parkinson\u2019s disease

Abnormal aggregation of misfolded pathological proteins in neurons is a prominent feature of neurodegenerative disorders including Parkinson’s disease (PD). Perturbations of proteostasis at the endoplasmic reticulum (ER) triggers ER stress, activating the unfolded protein response (UPR). Chronic ER stress is thought to underlie the death of neurons during the neurodegenerative progression, but the precise mechanism by which the UPR pathways regulate neuronal cell fate remains incompletely understood. Here we report a critical neurodegenerative role for inositol-requiring enzyme 1 (IRE1), the evolutionarily conserved ER stress sensor, in a Drosophila model of PD. We found that IRE1 was hyperactivated upon accumulation of α-synuclein in the fly photoreceptor neurons. Ectopic overexpression of IRE1 was sufficient to trigger autophagy-dependent neuron death in an XBP1-independent, JNK-dependent manner. Furthermore, IRE1 was able to promote dopaminergic neuron loss, progressive locomotor impairment, and shorter lifespan, whereas blocking IRE1 or ATG7 expression remarkably ameliorated the progression of α-synuclein-caused Parkinson’s disease. These results provide in vivo evidence demonstrating that the IRE1 pathway drives PD progression through coupling ER stress to autophagy-dependent neuron death.

Overexpression of Mitochondrial Calcium Uniporter Causes Neuronal Death.

Neurodegenerative diseases are a large and heterogeneous group of disorders characterized by selective and progressive death of specific neuronal subtypes. In most of the cases, the pathophysiology is still poorly understood, although a number of hypotheses have been proposed. Among these, dysregulation of Ca2+ homeostasis and mitochondrial dysfunction represent two broadly recognized early events associated with neurodegeneration. However, a direct link between these two hypotheses can be drawn. Mitochondria actively participate to global Ca2+ signaling, and increases of [Ca2+] inside organelle matrix are known to sustain energy production to modulate apoptosis and remodel cytosolic Ca2+ waves. Most importantly, while mitochondrial Ca2+ overload has been proposed as the no-return signal, triggering apoptotic or necrotic neuronal death, until now direct evidences supporting this hypothesis, especially in vivo, are limited. Here, we took advantage of the identification of the mitochondrial Ca2+ uniporter (MCU) and tested whether mitochondrial Ca2+ signaling controls neuronal cell fate. We overexpressed MCU both in vitro, in mouse primary cortical neurons, and in vivo, through stereotaxic injection of MCU-coding adenoviral particles in the brain cortex. We first measured mitochondrial Ca2+ uptake using quantitative genetically encoded Ca2+ probes, and we observed that the overexpression of MCU causes a dramatic increase of mitochondrial Ca2+ uptake both at resting and after membrane depolarization. MCU-mediated mitochondrial Ca2+ overload causes alteration of organelle morphology and dysregulation of global Ca2+ homeostasis. Most importantly, MCU overexpression in vivo is sufficient to trigger gliosis and neuronal loss. Overall, we demonstrated that mitochondrial Ca2+ overload is per se sufficient to cause neuronal cell death both in vitro and in vivo, thus highlighting a potential key step in neurodegeneration.

A Myt1 family transcription factor defines neuronal fate by repressing non-neuronal genes.

Cellular differentiation requires both activation of target cell transcriptional programs and repression of non-target cell programs. The Myt1 family of zinc finger transcription factors contributes to fibroblast to neuron reprogramming in vitro. Here, we show that ztf-11 (Zinc-finger Transcription Factor-11), the sole Caenorhabditis elegans Myt1 homolog, is required for neurogenesis in multiple neuronal lineages from previously differentiated epithelial cells, including a neuron generated by a developmental epithelial-to-neuronal transdifferentiation event. ztf-11 is exclusively expressed in all neuronal precursors with remarkable specificity at single-cell resolution. Loss of ztf-11 leads to upregulation of non-neuronal genes and reduced neurogenesis. Ectopic expression of ztf-11 in epidermal lineages is sufficient to produce additional neurons. ZTF-11 functions together with the MuvB corepressor complex to suppress the activation of non-neuronal genes in neurons. These results dovetail with the ability of Myt1l (Myt1-like) to drive neuronal transdifferentiation in vitro in vertebrate systems. Together, we identified an evolutionarily conserved mechanism to specify neuronal cell fate by repressing non-neuronal genes.

Abstract 83: The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer

Abstract There will be an estimated 1.8 million new cancer diagnoses in the US this year alone, with >80% of these cases driven by oncogenic K-RAS/SIAH pathway activation, even in the absence of oncogenic RAS mutations. Over three decades of intense research has focused on conquering RAS pathway activation in cancer, yet oncogenic K-RAS has remained essentially undruggable. Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway thus far. Underscoring SINA’s importance is its high degree of evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung, and breast cancers. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the Drosophila photoreceptor development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, and several newly identified point mutant alleles exhibit stronger mutant phenotypes than those of previously published truncated alleles. Sequencing analysis of these sinamutant revealed critical roles of several immutable amino acid residues indispensable for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we generated a collection of transgenic fly lines that carry either wild-type (WT) or dominant negative (DN) DmSINA and human SIAH1/2. Tissue-specific SINA/SIAH expression using GAL4 drivers revealed the biological consequences of gain or loss of function of SINA/SIAH1/2 in transmitting RAS signaling in Drosophila development. Immunofluorescent (IF) staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted dramatic changes in neuronal cell fate in the developing eye and sensory neuronal cells on the notum. Furthermore, IF staining of larval salivary glands revealed a critical role for SINA/SIAH in regulating cell shape, focal adhesions, and cellular junctions. Results from these transgenic models suggest that Drosophila SINA and human SIAH1/2 are highly evolutionarily conserved and functionally interchangeable. Ultimately, we aim to translate these preclinical findings to demonstrate the high potency and anticancer efficacy of an anti-SIAH-based anti-K-RAS strategy against multidrug-resistant and incurable human cancer. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 83.

Insulin/IGF Signaling and Vitellogenin Provisioning Mediate Intergenerational Adaptation to Nutrient Stress

The roundworm C.elegans reversibly arrests larval development during starvation [1], but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success [4]. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning ofvitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.

Quantitative single-cell live imaging links HES5 dynamics with cell-state and fate in murine neurogenesis

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.

Microglia activation induces oxidative injury and decreases SIRT3 expression in dopaminergic neuronal cells.

Microglia activation-mediated neuroinflammation plays an important role in the progression of Parkinson's disease (PD). However, effects of microglia activation on dopaminergic neuronal cell (DAC) fate are still poorly understood. The objective of this study was to explore the neurotoxic effects of microglia activation-mediated oxidative injury in DACs and its possible mechanisms. In the present study, microglia-DACs co-culture systems (murine BV-2 and MN9D cells, or primary microglia and mesencephalic neurons) were used to display the crosstalk between both cell types. The cytotoxicity of lipopolysaccharide-induced microglia activation led to the accumulation of intracellular reactive oxygen species, increased cell apoptosis rate, reduced number of DACs, concomitant to cell cycle arrest at G1 phase. Molecular mechanisms of apoptosis caused by microglia activation-induced oxidative injury included the increased opening of mitochondrial permeability transition pore and enhanced membrane potential depolarization in MN9D cells, down-regulation of Bcl-2 and up-regulation of Bax, caspase-3 expression in DACs. In addition, microglia activation made a significant reduction of SIRT3 and superoxide dismutase 2 gene expression in DACs. Taken together, these data imply that microglia activation promotes cell apoptosis through mitochondrial pathway and decreases SIRT3 expression in DACs, which may provide some support for PD progression promoted by neuroinflammation.

A branching gene regulatory network dictating different aspects of a neuronal cell identity.

The nervous system displays a daunting cellular diversity. Neuronal subtypes differ from each other in several aspects, including their neurotransmitter expression and axon projection. These aspects can converge, but can also diverge, such that neurons expressing the same neurotransmitter may project axons to different targets. It is not well understood how regulatory programs converge/diverge to associate/dissociate different cell fate features. Studies of the Drosophila Tv1 neurons have identified a regulatory cascade, ladybird early→collier→apterous/eyes absent→dimmed, that specifies Tv1 neurotransmitter expression. Here, we conduct genetic and transcriptome analysis to address how other aspects of Tv1 cell fate are governed. We find that an initiator terminal selector gene triggers a feedforward loop that branches into different subroutines, each of which establishes different features of this one unique neuronal cell fate.

Inhibition of TNF-α-induced neuronal apoptosis by antidepressants acting through the lysophosphatidic acid receptor LPA1.

Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine considered to be implicated in the pathogenesis of major depressive disorder, is a critical regulator of neuronal cell fate. In the present study we found that TNF-α-induced apoptosis of HT22 hippocampal cells, a neuroblast-like cell line, was markedly attenuated by the antidepressants mianserin, mirtazapine and amitriptyline. The anti-apoptotic effect of the antidepressants was blocked by either pharmacological inhibition or gene silencing of the lysophosphatidic acid receptor LPA1. Mianserin failed to affect TNF-α-induced caspase 8 activation, but inhibited the loss of mitochondrial membrane potential, the release of cytochrome c from mitochondria, procaspase 9 cleavage and downstream activation of caspase 3 in response to the cytokine. By acting through LPA1, mianserin also attenuated the enhanced pro-apoptotic response induced by the combination of TNF-α with other pro-inflammatory cytokines. TNF-α appeared to counterbalance its own pro-apoptotic response by activating NF-kB, ERK1/2 and JNK. Antidepressants had no significant effects on NF-kB activation, but potentiated the TAK-1-dependent phosphorylation of ERK1/2 and JNK elicited by the cytokine. This synergistic interaction was associated with enhanced JNK-mediated phosphorylation of Bcl-2at Ser70 and increased ERK1/2-dependent mitochondrial accumulation of Mcl-1, two anti-apoptotic proteins that promote mitochondrial outer membrane stability. These results indicate that certain antidepressants, by activating LPA1 signalling, protect HT22 hippocampal cells from TNF-α-induced apoptosis through a mechanism involving, at least in part, the potentiation of the pro-survival pathways activated by the cytokine.

Altered expression of dopaminergic cell fate regulating genes prior to manifestation of symptoms in a transgenic rat model of Huntington's disease

Hyperactivity of the dopaminergic pathway is thought to contribute to clinical symptoms in the early stages of Huntington’s disease (HD). It is suggested to be result of a reduced dopaminergic inhibition by degeneration of medium spiny neurons in the striatum.Previously, we have shown that the number of dopaminergic cells is increased in the dorsal raphe nucleus (DRN) of HD patients and transgenic HD (tgHD) rats during the manifestation phase of the disease; as well as in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) of tgHD rats. To address whether these changes are secondary to neurodegeneration or take place in the pre-manifest phase of the disease, we examined the expression of genes controlling neuronal cell fate and genes that define dopaminergic cell phenotype.In the SNc-VTA of tgHD rats, Msx1 was upregulated, which correlated with an altered expression of transcription factors Zbtb16 and Tcf12. Zbtb16 was upregulated in the DRN and it was the only gene that showed a correlated expression in the tgHD rats between SNc-VTA and DRN. Zbtb16 may be a candidate for regionally tuning its cell populations, resulting in the increase in dopaminergic cells observed in our previous studies.Here, we demonstrated an altered expression of genes related to dopaminergic cell fate regulation in the brainstem of 6 months-old tgHD rats. This suggests that changes in dopaminergic system in HD precede the manifestation of clinical symptoms, contradicting the theory that hyperdopaminergic status in HD is a consequence of neurodegeneration in the striatum.

Transcription factor Ptf1a in development, diseases and reprogramming.

The transcription factor Ptf1a is a crucial helix-loop-helix (bHLH) protein selectively expressed in the pancreas, retina, spinal cord, brain, and enteric nervous system. Ptf1a is preferably assembled into a transcription trimeric complex PTF1 with an E protein and Rbpj (or Rbpjl). In pancreatic development, Ptf1a is indispensable in controlling the expansion of multipotent progenitor cells as well as the specification and maintenance of the acinar cells. In neural tissues, Ptf1a is transiently expressed in the post-mitotic cells and specifies the inhibitory neuronal cell fates, mostly mediated by downstream genes such as Tfap2a/b and Prdm13. Mutations in the coding and non-coding regulatory sequences resulting in Ptf1a gain- or loss-of-function are associated with genetic diseases such as pancreatic and cerebellar agenesis in the rodent and human. Surprisingly, Ptf1a alone is sufficient to reprogram mouse or human fibroblasts into tripotential neural stem cells. Its pleiotropic functions in many biological processes remain to be deciphered in the future.

The RNA-binding protein LIN28 controls progenitor and neuronal cell fate during postnatal neurogenesis.

The RNA-binding protein LIN28 is known to regulate cell fate, tissue growth, and pluripotency; however, a unified understanding of its role at the cellular level has not been achieved. Here, we address its developmental activity in mammalian postnatal neurogenesis. Constitutive expression of LIN28 in progenitor cells of the mouse subventricular zone (SVZ) caused several distinct effects: 1) the number of differentiated neurons in the olfactory bulb was dramatically reduced, whereas the relative abundance of 2 neuronal subtypes was significantly altered, 2) the population of proliferating neural progenitors in the SVZ was reduced, whereas the proportion of neuroblasts was increased, and 3) the number of astrocytes was reduced, occasionally causing them to appear early. Thus, LIN28 acts at a poststem cell/predifferentiation step, and its continuous expression caused a precocious phenotype unlike in other experimental systems. Furthermore, for the first time in a vertebrate system, we separate the majority of the biologic role of LIN28 from its known activity of blocking the microRNA let-7 by using a circular RNA sponge. We find that although LIN28 has a multifaceted role in the number and types of cells produced during postnatal neurogenesis, it appears that its action through let-7 is responsible for only a fraction of these effects.—Romer-Seibert, J. S., Hartman, N. W., Moss, E. G. The RNA-binding protein LIN28 controls progenitor and neuronal cell fate during postnatal neurogenesis.

The Scalloped and Nerfin-1 Transcription Factors Cooperate to Maintain Neuronal Cell Fate

The ability of cells to stably maintain their fate is governed by specific transcription regulators. Here, we show that the Scalloped (Sd) and Nervous fingers-1 (Nerfin-1) transcription factors physically and functionally interact to maintain medulla neuron fate in the Drosophila melanogaster CNS. Using Targeted DamID, we find that Sd and Nerfin-1 occupy a highly overlapping set of target genes, including regulators of neural stem cell and neuron fate, and signaling pathways that regulate CNS development such as Notch and Hippo. Modulation of either Sd or Nerfin-1 activity causes medulla neurons to dedifferentiate toa stem cell-like state, and this is mediated at least in part by Notch pathway deregulation. Intriguingly, orthologs of Sd and Nerfin-1 have also been implicated in control of neuronal cell fate decisions in both worms and mammals. Our data indicate that this transcription factor pair exhibits remarkable biochemical and functional conservation across metazoans.

Specification of Drosophila neuropeptidergic neurons by the splicing component brr2.

During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. In this study, we focus on the role of RNA processing during target-derived signaling. We found that brr2 and Prp8 play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system.

Glycans and glycosaminoglycans in neurobiology: key regulators of neuronal cell function and fate.

The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin sulfate/dermatan sulfate (CS/DS) with selected functional molecules in neural tissues. Cell surface glycans and GAGs have evolved over millions of years to become cellular mediators which regulate fundamental aspects of cellular survival. The glycocalyx, which surrounds all cells, actuates responses to growth factors, cytokines and morphogens at the cellular boundary, silencing or activating downstream signaling pathways and gene expression. In this review, we have focused on interactions mediated by l-fucose, KS and CS/DS in the central and peripheral nervous systems. Fucose makes critical contributions in the area of molecular recognition and information transfer in the blood group substances, cytotoxic immunoglobulins, cell fate-mediated Notch-1 interactions, regulation of selectin-mediated neutrophil extravasation in innate immunity and CD-34-mediated new blood vessel development, and the targeting of neuroprogenitor cells to damaged neural tissue. Fucosylated glycoproteins regulate delivery of synaptic neurotransmitters and neural function. Neural KS proteoglycans (PGs) were examined in terms of cellular regulation and their interactive properties with neuroregulatory molecules. The paradoxical properties of CS/DS isomers decorating matrix and transmembrane PGs and the positive and negative regulatory cues they provide to neurons are also discussed.