Publications by authors named "Douglas W Allan"

25 Publications

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A low affinity -regulatory BMP response element restricts target gene activation to subsets of Drosophila neurons.

Elife 2020 10 30;9. Epub 2020 Oct 30.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

Retrograde BMP signaling and canonical pMad/Medea-mediated transcription regulate diverse target genes across subsets of efferent neurons, to differentiate neuropeptidergic neurons and promote motor neuron terminal maturation. How a common BMP signal regulates diverse target genes across many neuronal subsets remains largely unresolved, although available evidence implicates subset-specific transcription factor codes rather than differences in BMP signaling. Here we examine the regulatory mechanisms restricting BMP-induced neuropeptide expression to Tv4-neurons. We find that pMad/Medea bind at an atypical, low affinity motif in the enhancer. Converting this motif to high affinity caused ectopic enhancer activity and eliminated Tv4-neuron expression. In silico searches identified additional motif instances functional in other efferent neurons, implicating broader functions for this motif in BMP-dependent enhancer activity. Thus, differential interpretation of a common BMP signal, conferred by low affinity pMad/Medea binding motifs, can contribute to the specification of BMP target genes in efferent neuron subsets.
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http://dx.doi.org/10.7554/eLife.59650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669266PMC
October 2020

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Nat Commun 2020 04 29;11(1):2073. Epub 2020 Apr 29.

Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, Canada.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.
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http://dx.doi.org/10.1038/s41467-020-15943-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190743PMC
April 2020

Retrograde BMP signaling activates neuronal gene expression through widespread deployment of a conserved BMP-responsive cis-regulatory activation element.

Nucleic Acids Res 2019 01;47(2):679-699

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Retrograde Bone Morphogenetic Protein (BMP) signaling in neurons is essential for the differentiation and synaptic function of many neuronal subtypes. BMP signaling regulates these processes via Smad transcription factor activity, yet the scope and nature of Smad-dependent gene regulation in neurons are mostly unknown. Here, we applied a computational approach to predict Smad-binding cis-regulatory BMP-Activating Elements (BMP-AEs) in Drosophila, followed by transgenic in vivo reporter analysis to test their neuronal subtype enhancer activity in the larval central nervous system (CNS). We identified 34 BMP-AE-containing genomic fragments that are responsive to BMP signaling in neurons, and showed that the embedded BMP-AEs are required for this activity. RNA-seq analysis identified BMP-responsive genes in the CNS and revealed that BMP-AEs selectively enrich near BMP-activated genes. These data suggest that functional BMP-AEs control nearby BMP-activated genes, which we validated experimentally. Finally, we demonstrated that the BMP-AE motif mediates a conserved Smad-responsive function in the Drosophila and vertebrate CNS. Our results provide evidence that BMP signaling controls neuronal function by directly coordinating the expression of a battery of genes through widespread deployment of a conserved Smad-responsive cis-regulatory motif.
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http://dx.doi.org/10.1093/nar/gky1135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344883PMC
January 2019

IRES-dependent ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection.

Nucleic Acids Res 2018 12;46(22):11952-11967

Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

RNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses 37 nucleotides downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.
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http://dx.doi.org/10.1093/nar/gky1121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294563PMC
December 2018

Segmentally homologous neurons acquire two different terminal neuropeptidergic fates in the Drosophila nervous system.

PLoS One 2018 10;13(4):e0194281. Epub 2018 Apr 10.

Departamento de Biología, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

In this study, we identify the means by which segmentally homologous neurons acquire different neuropeptide fates in Drosophila. Ventral abdominal (Va)-neurons in the A1 segment of the ventral nerve cord express DH31 and AstA neuropeptides (neuropeptidergic fate I) by virtue of Ubx activity, whereas the A2-A4 Va-neurons express the Capa neuropeptide (neuropeptidergic fate II) under the influence of abdA. These different fates are attained through segment-specific programs of neural subtype specification undergone by segmentally homologous neurons. This is an attractive alternative by which Hox genes can shape Drosophila segmental neural architecture (more sophisticated than the previously identified binary "to live" or "not to live" mechanism). These data refine our knowledge of the mechanisms involved in diversifying neuronal identity within the central nervous system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194281PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892886PMC
July 2018

female-specific Ilp7 motoneurons are generated by Fruitless-dependent cell death in males and by a double-assurance survival role for Transformer in females.

Development 2018 01 8;145(1). Epub 2018 Jan 8.

Department of Cellular and Physiological Sciences, University of British Columbia, 2420 Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada

Female-specific Ilp7 neuropeptide-expressing motoneurons (FS-Ilp7 motoneurons) are required in for oviduct function in egg laying. Here, we uncover cellular and genetic mechanisms underlying their female-specific generation. We demonstrate that programmed cell death (PCD) eliminates FS-Ilp7 motoneurons in males, and that this requires male-specific splicing of the sex-determination gene () into the Fru isoform. However, in females, alleles that only generate Fru isoforms failed to kill FS-Ilp7 motoneurons. This blockade of Fru-dependent PCD was not attributable to gene function but to a non-canonical role for (), a gene encoding the RNA splicing activator that regulates female-specific splicing of and transcripts. In both sexes, we show that Tra prevents PCD even when the Fru isoform is expressed. In addition, we found that Fru eliminated FS-Ilp7 motoneurons in both sexes, but only when Tra was absent. Thus, Fru-dependent PCD eliminates female-specific neurons in males, and Tra plays a double-assurance function in females to establish and reinforce the decision to generate female-specific neurons.
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http://dx.doi.org/10.1242/dev.150821DOI Listing
January 2018

acts in the PLK1-dependent spindle positioning pathway and supports neural development.

Elife 2017 10 10;6. Epub 2017 Oct 10.

Department of Paediatrics, University of British Columbia, Vancouver, Canada.

Oriented cell division is one mechanism progenitor cells use during development and to maintain tissue homeostasis. Common to most cell types is the asymmetric establishment and regulation of cortical NuMA-dynein complexes that position the mitotic spindle. Here, we discover that HMMR acts at centrosomes in a PLK1-dependent pathway that locates active Ran and modulates the cortical localization of NuMA-dynein complexes to correct mispositioned spindles. This pathway was discovered through the creation and analysis of -knockout mice, which suffer neonatal lethality with defective neural development and pleiotropic phenotypes in multiple tissues. HMMR over-expression in immortalized cancer cells induces phenotypes consistent with an increase in active Ran including defects in spindle orientation. These data identify an essential role for HMMR in the PLK1-dependent regulatory pathway that orients progenitor cell division and supports neural development.
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http://dx.doi.org/10.7554/eLife.28672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681225PMC
October 2017

The Channel Gene Promotes Synaptic Transmission and Coordinated Movement in .

Front Mol Neurosci 2017 24;10:41. Epub 2017 Feb 24.

Department of Cellular and Physiological Sciences, The University of British Columbia Vancouver, BC, Canada.

Hyperpolarization-activated cyclic nucleotide-gated "HCN" channels, which underlie the hyperpolarization-activated current (I), have been proposed to play diverse roles in neurons. The presynaptic HCN channel is thought to both promote and inhibit neurotransmitter release from synapses, depending upon its interactions with other presynaptic ion channels. In larvae of , inhibition of the presynaptic HCN channel by the drug ZD7288 reduces the enhancement of neurotransmitter release at motor terminals by serotonin but this drug has no effect on basal neurotransmitter release, implying that the channel does not contribute to firing under basal conditions. Here, we show that genetic disruption of the sole HCN gene reduces the amplitude of the evoked response at the neuromuscular junction (NMJ) of third instar larvae by decreasing the number of released vesicles. The anatomy of the (NMJ) is not notably affected by disruption of the gene. We propose that the presynaptic HCN channel is active under basal conditions and promotes neurotransmission at larval motor terminals. Finally, we demonstrate that partial loss-of-function mutant adult flies have impaired locomotion, and, thus, we hypothesize that the presynaptic HCN channel at the (NMJ) may contribute to coordinated movement.
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http://dx.doi.org/10.3389/fnmol.2017.00041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323408PMC
February 2017

Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein.

J Virol 2017 03 14;91(5). Epub 2017 Feb 14.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada

Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication. RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.
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http://dx.doi.org/10.1128/JVI.01779-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309961PMC
March 2017

Gene Regulatory Mechanisms Underlying the Spatial and Temporal Regulation of Target-Dependent Gene Expression in Drosophila Neurons.

PLoS Genet 2015 Dec 29;11(12):e1005754. Epub 2015 Dec 29.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Neuronal differentiation often requires target-derived signals from the cells they innervate. These signals typically activate neural subtype-specific genes, but the gene regulatory mechanisms remain largely unknown. Highly restricted expression of the FMRFa neuropeptide in Drosophila Tv4 neurons requires target-derived BMP signaling and a transcription factor code that includes Apterous. Using integrase transgenesis of enhancer reporters, we functionally dissected the Tv4-enhancer of FMRFa within its native cellular context. We identified two essential but discrete cis-elements, a BMP-response element (BMP-RE) that binds BMP-activated pMad, and a homeodomain-response element (HD-RE) that binds Apterous. These cis-elements have low activity and must be combined for Tv4-enhancer activity. Such combinatorial activity is often a mechanism for restricting expression to the intersection of cis-element spatiotemporal activities. However, concatemers of the HD-RE and BMP-RE cis-elements were found to independently generate the same spatiotemporal expression as the Tv4-enhancer. Thus, the Tv4-enhancer atypically combines two low-activity cis-elements that confer the same output from distinct inputs. The activation of target-dependent genes is assumed to 'wait' for target contact. We tested this directly, and unexpectedly found that premature BMP activity could not induce early FMRFa expression; also, we show that the BMP-insensitive HD-RE cis-element is activated at the time of target contact. This led us to uncover a role for the nuclear receptor, seven up (svp), as a repressor of FMRFa induction prior to target contact. Svp is normally downregulated immediately prior to target contact, and we found that maintaining Svp expression prevents cis-element activation, whereas reducing svp gene dosage prematurely activates cis-element activity. We conclude that the target-dependent FMRFa gene is repressed prior to target contact, and that target-derived BMP signaling directly activates FMRFa gene expression through an atypical gene regulatory mechanism.
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http://dx.doi.org/10.1371/journal.pgen.1005754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694770PMC
December 2015

Rescue from tau-induced neuronal dysfunction produces insoluble tau oligomers.

Sci Rep 2015 Nov 26;5:17191. Epub 2015 Nov 26.

Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.

Aggregation of highly phosphorylated tau is a hallmark of Alzheimer's disease and other tauopathies. Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase glycogen-synthase-kinase-3beta (GSK-3β). Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure. This may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others. Whether these are inert or actively protective remains to be established. Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.
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http://dx.doi.org/10.1038/srep17191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660438PMC
November 2015

Transcriptional selectors, masters, and combinatorial codes: regulatory principles of neural subtype specification.

Wiley Interdiscip Rev Dev Biol 2015 Sep-Oct;4(5):505-28. Epub 2015 Apr 8.

Department of Clinical and Experimental Medicine, Linkoping University, Linkoping, Sweden.

The broad range of tissue and cellular diversity of animals is generated to a large extent by the hierarchical deployment of sequence-specific transcription factors and co-factors (collectively referred to as TF's herein) during development. Our understanding of these developmental processes has been facilitated by the recognition that the activities of many TF's can be meaningfully described by a few functional categories that usefully convey a sense for how the TF's function, and also provides a sense for the regulatory organization of the developmental processes in which they participate. Here, we draw on examples from studies in Caenorhabditis elegans, Drosophila melanogaster, and vertebrates to discuss how the terms spatial selector, temporal selector, tissue/cell type selector, terminal selector and combinatorial code may be usefully applied to categorize the activities of TF's at critical steps of nervous system construction. While we believe that these functional categories are useful for understanding the organizational principles by which TF's direct nervous system construction, we however caution against the assumption that a TF's function can be solely or fully defined by any single functional category. Indeed, most TF's play diverse roles within different functional categories, and their roles can blur the lines we draw between these categories. Regardless, it is our belief that the concepts discussed here are helpful in clarifying the regulatory complexities of nervous system development, and hope they prove useful when interpreting mutant phenotypes, designing future experiments, and programming specific neuronal cell types for use in therapies.
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http://dx.doi.org/10.1002/wdev.191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672696PMC
May 2016

Homeotic function of Drosophila Bithorax-complex miRNAs mediates fertility by restricting multiple Hox genes and TALE cofactors in the CNS.

Dev Cell 2014 Jun 5;29(6):635-48. Epub 2014 Jun 5.

Department of Developmental Biology, Sloan-Kettering Institute, 1275 York Avenue, Box 252, New York, NY 10065, USA. Electronic address:

The Drosophila Bithorax complex (BX-C) Hox cluster contains a bidirectionally transcribed miRNA locus, and a deletion mutant (Δmir) lays no eggs and is completely sterile. We show these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the CNS. Δmir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors. These are phenotypically critical targets, because sterility of Δmir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in Δmir females, and substantially rescued by heterozygosity of Δmir targets, especially within the BX-C. Collectively, we reveal (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation; and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior.
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http://dx.doi.org/10.1016/j.devcel.2014.04.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111139PMC
June 2014

Female-biased dimorphism underlies a female-specific role for post-embryonic Ilp7 neurons in Drosophila fertility.

Development 2013 Sep;140(18):3915-26

Department of Cellular and Physiological Sciences, 2401 Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

In Drosophila melanogaster, much of our understanding of sexually dimorphic neuronal development and function comes from the study of male behavior, leaving female behavior less well understood. Here, we identify a post-embryonic population of Insulin-like peptide 7 (Ilp7)-expressing neurons in the posterior ventral nerve cord that innervate the reproductive tracts and exhibit a female bias in their function. They form two distinct dorsal and ventral subsets in females, but only a single dorsal subset in males, signifying a rare example of a female-specific neuronal subset. Female post-embryonic Ilp7 neurons are glutamatergic motoneurons innervating the oviduct and are required for female fertility. In males, they are serotonergic/glutamatergic neuromodulatory neurons innervating the seminal vesicle but are not required for male fertility. In both sexes, these neurons express the sex-differentially spliced fruitless-P1 transcript but not doublesex. The male fruitless-P1 isoform (fruM) was necessary and sufficient for serotonin expression in the shared dorsal Ilp7 subset, but although it was necessary for eliminating female-specific Ilp7 neurons in males, it was not sufficient for their elimination in females. By contrast, sex-specific RNA-splicing by female-specific transformer is necessary for female-type Ilp7 neurons in females and is sufficient for their induction in males. Thus, the emergence of female-biased post-embryonic Ilp7 neurons is mediated in a subset-specific manner by a tra- and fru-dependent mechanism in the shared dorsal subset, and a tra-dependent, fru-independent mechanism in the female-specific subset. These studies provide an important counterpoint to studies of the development and function of male-biased neuronal dimorphism in Drosophila.
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http://dx.doi.org/10.1242/dev.094714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3915572PMC
September 2013

Subtype-specific neuronal remodeling during Drosophila metamorphosis.

Fly (Austin) 2013 Apr-Jun;7(2):78-86. Epub 2013 Apr 1.

Department of Cellular and Physiological Sciences, Life Sciences Centre, Health Sciences Mall, University of British Columbia, Vancouver, BC Canada.

During metamorphosis in holometabolous insects, the nervous system undergoes dramatic remodeling as it transitions from its larval to its adult form. Many neurons are generated through post-embryonic neurogenesis to have adult-specific roles, but perhaps more striking is the dramatic remodeling that occurs to transition neurons from functioning in the larval to the adult nervous system. These neurons exhibit a remarkable degree of plasticity during this transition; many subsets undergo programmed cell death, others remodel their axonal and dendritic arbors extensively, whereas others undergo trans-differentiation to alter their terminal differentiation gene expression profiles. Yet other neurons appear to be developmentally frozen in an immature state throughout larval life, to be awakened at metamorphosis by a process we term temporally-tuned differentiation. These multiple forms of remodeling arise from subtype-specific responses to a single metamorphic trigger, ecdysone. Here, we discuss recent progress in Drosophila melanogaster that is shedding light on how subtype-specific programs of neuronal remodeling are generated during metamorphosis.
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http://dx.doi.org/10.4161/fly.23969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732335PMC
January 2014

Maintenance of β-cell maturity and plasticity in the adult pancreas: developmental biology concepts in adult physiology.

Diabetes 2012 Jun;61(6):1365-71

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

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http://dx.doi.org/10.2337/db11-1361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357305PMC
June 2012

Temporally tuned neuronal differentiation supports the functional remodeling of a neuronal network in Drosophila.

Proc Natl Acad Sci U S A 2012 Mar 5;109(13):E748-56. Epub 2012 Mar 5.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.

During insect metamorphosis, neuronal networks undergo extensive remodeling by restructuring their connectivity and recruiting newborn neurons from postembryonic lineages. The neuronal network that directs the essential behavior, ecdysis, generates a distinct behavioral sequence at each developmental transition. Larval ecdysis replaces the cuticle between larval stages, and pupal ecdysis externalizes and expands the head and appendages to their adult position. However, the network changes that support these differences are unknown. Crustacean cardioactive peptide (CCAP) neurons and the peptide hormones they secrete are critical for ecdysis; their targeted ablation alters larval ecdysis progression and results in a failure of pupal ecdysis. In this study, we demonstrate that the CCAP neuron network is remodeled immediately before pupal ecdysis by the emergence of 12 late CCAP neurons. All 12 are CCAP efferents that exit the central nervous system. Importantly, these late CCAP neurons were found to be entirely sufficient for wild-type pupal ecdysis, even after targeted ablation of all other 42 CCAP neurons. Our evidence indicates that late CCAP neurons are derived from early, likely embryonic, lineages. However, they do not differentiate to express their peptide hormone battery, nor do they project an axon via lateral nerve trunks until pupariation, both of which are believed to be critical for the function of CCAP efferent neurons in ecdysis. Further analysis implicated ecdysone signaling via ecdysone receptors A/B1 and the nuclear receptor ftz-f1 as the differentiation trigger. These results demonstrate the utility of temporally tuned neuronal differentiation as a hard-wired developmental mechanism to remodel a neuronal network to generate a scheduled change in behavior.
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http://dx.doi.org/10.1073/pnas.1114710109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324015PMC
March 2012

Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system.

PLoS Genet 2012 23;8(2):e1002501. Epub 2012 Feb 23.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply "locked-in," but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain.
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http://dx.doi.org/10.1371/journal.pgen.1002501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285578PMC
February 2013

Retrograde BMP signaling controls Drosophila behavior through regulation of a peptide hormone battery.

Development 2011 Aug;138(15):3147-57

Department of Cellular and Physiological Sciences, Room 2420 Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

Retrograde BMP signaling in neurons plays conserved roles in synaptic efficacy and subtype-specific gene expression. However, a role for retrograde BMP signaling in the behavioral output of neuronal networks has not been established. Insect development proceeds through a series of stages punctuated by ecdysis, a complex patterned behavior coordinated by a dedicated neuronal network. In Drosophila, larval ecdysis sheds the old cuticle between larval stages, and pupal ecdysis everts the head and appendages to their adult external position during metamorphosis. Here, we found that mutants of the type II BMP receptor wit exhibited a defect in the timing of larval ecdysis and in the completion of pupal ecdysis. These phenotypes largely recapitulate those previously observed upon ablation of CCAP neurons, an integral subset of the ecdysis neuronal network. Here, we establish that retrograde BMP signaling in only the efferent subset of CCAP neurons (CCAP-ENs) is required to cell-autonomously upregulate expression of the peptide hormones CCAP, Mip and Bursicon β. In wit mutants, restoration of wit exclusively in CCAP neurons significantly rescued peptide hormone expression and ecdysis phenotypes. Moreover, combinatorial restoration of peptide hormone expression in CCAP neurons in wit mutants also significantly rescued wit ecdysis phenotypes. Collectively, our data demonstrate a novel role for retrograde BMP signaling in maintaining the behavioral output of a neuronal network and uncover the underlying cellular and gene regulatory substrates.
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http://dx.doi.org/10.1242/dev.064105DOI Listing
August 2011

Neuronal phenotype in the mature nervous system is maintained by persistent retrograde bone morphogenetic protein signaling.

J Neurosci 2009 Mar;29(12):3852-64

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

The terminal differentiation of many developing neurons occurs after they innervate their target cells and is triggered by secreted target-derived signals that are transduced by presynaptic cognate receptors. Such retrograde signaling induces the expression of genes that are often distinctive markers of neuronal phenotype and function. However, whether long-term maintenance of neuronal phenotype requires persistent retrograde signaling remains poorly understood. Previously, we demonstrated that retrograde bone morphogenetic protein (BMP) signaling induces expression of a phenotypic marker of Drosophila Tv neurons, the neuropeptide FMRFamide (FMRFa). Here, we used a genetic technique that spatiotemporally targets transgene expression in Drosophila to test the role of persistent BMP signaling in the maintenance of Tv phenotype. We show that expression of dominant blockers of BMP signaling selectively in adult Tv neurons dramatically downregulated FMRFa expression. Moreover, adult-onset expression of mutant Glued, which blocks dynein/dynactin-mediated retrograde axonal transport, eliminated retrograde BMP signaling and dramatically downregulated FMRFa expression. Finally, we found that BMP deprivation did not affect Tv neuron survival and that FMRFa expression fully recovered to control levels after the termination of BMP blockade or Glued expression. Our results show that persistent retrograde BMP signaling is required to induce and to subsequently maintain the expression of a stably expressed phenotypic marker in a subset of mature Drosophila neurons. We postulate that retrograde maintenance of neuronal phenotype is conserved in vertebrates, and as a consequence, neuronal phenotype is likely vulnerable to neurodegenerative disease pathologies that disrupt neuronal connectivity or axonal transport.
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http://dx.doi.org/10.1523/JNEUROSCI.0213-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6665019PMC
March 2009

Regulators acting in combinatorial codes also act independently in single differentiating neurons.

Neuron 2005 Mar;45(5):689-700

Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, we have resolved how each of these factors contributes to ap neuron diversity. We find that these factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, we also find that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, we show that single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits.
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http://dx.doi.org/10.1016/j.neuron.2005.01.026DOI Listing
March 2005

Independent roles of the dachshund and eyes absent genes in BMP signaling, axon pathfinding and neuronal specification.

Development 2004 Dec 3;131(23):5837-48. Epub 2004 Nov 3.

Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

In the Drosophila nerve cord, a subset of neurons expresses the neuropeptide FMRFamide related (Fmrf). Fmrf expression is controlled by a combinatorial code of intrinsic factors and an extrinsic BMP signal. However, this previously identified code does not fully explain the regulation of Fmrf. We have found that the Dachshund (Dac) and Eyes Absent (Eya) transcription co-factors participate in this combinatorial code. Previous studies have revealed an intimate link between Dac and Eya during eye development. Here, by analyzing their function in neurons with multiple phenotypic markers, we demonstrate that they play independent roles in neuronal specification, even within single cells. dac is required for high-level Fmrf expression, and acts potently together with apterous and BMP signaling to trigger Fmrf expression ectopically, even in motoneurons. By contrast, eya regulates Fmrf expression by controlling both axon pathfinding and BMP signaling, but cannot trigger Fmrf ectopically. Thus, we show that dac and eya perform entirely different functions in a single cell type to ultimately regulate a single phenotypic outcome.
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http://dx.doi.org/10.1242/dev.01447DOI Listing
December 2004

Together at last: bHLH and LIM-HD regulators cooperate to specify motor neurons.

Neuron 2003 Jun;38(5):675-7

Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

bHLH and LIM-HD transcription factors were originally thought to act at different stages of neurogenesis: bHLHs as neuronal "inducers" and LIM-HDs as postmitotic subtype determinants. These distinctions are becoming blurred, and a current study by Lee and Pfaff in this issue of Neuron shows that interaction between these factors functions to synchronize neurogenesis with neuronal cell type specification.
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http://dx.doi.org/10.1016/s0896-6273(03)00329-5DOI Listing
June 2003

Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code.

Cell 2003 Apr;113(1):73-86

Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord, the six Tv neurons express the neuropeptide gene FMRFamide. Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain gene apterous. We find that the zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this "code," by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells.
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http://dx.doi.org/10.1016/s0092-8674(03)00204-6DOI Listing
April 2003

Embryological origins and development of the rat diaphragm.

J Comp Neurol 2003 Jan;455(4):477-87

Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.

Textbooks of embryology provide a standard set of drawings and text reflecting the traditional interpretation of phrenic nerve and diaphragm development based on anatomical dissections of embryonic tissue. Here, we revisit this issue, taking advantage of immunohistochemical markers for muscle precursors in conjunction with mouse mutants to perform a systematic examination of phrenic-diaphragm embryogenesis. This includes examining the spatiotemporal relationship of phrenic axon outgrowth and muscle precursors during different stages of myogenesis. Additionally, mutant mice lacking c-met receptors were used to visualize the mesenchymal substratum of the developing diaphragm in the absence of myogenic cells. We found no evidence for contributions to the diaphragm musculature from the lateral body wall, septum transversum, or esophageal mesenchyme, as standard dogma would state. Nor did the data support the hypothesis that the crural diaphragm is of distinct embryological origins. Rather, we found that myogenic cells and axons destined to form the neuromuscular component of the diaphragm coalesce within the pleuroperitoneal fold (PPF). It is the expansion of these components of the PPF that leads to the formation of the diaphragm. Furthermore, we extended these studies to examine the developing diaphragm in an animal model of congenital diaphragmatic hernia (CDH). We find that malformation of the PPF mesenchymal substratum leads to the defect characteristic of CDH. In summary, the data demonstrates that a significant revision of narratives describing normal and pathological development of the diaphragm is warranted.
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http://dx.doi.org/10.1002/cne.10503DOI Listing
January 2003