scholarly journals A Protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development

2021 ◽  
Author(s):  
Deepika Vasudevan ◽  
Hidetaka Katow ◽  
Huai-Wei Huang ◽  
Grace Tang ◽  
Hyung Don Ryoo
Keyword(s):  
Control Mechanisms ◽  
Loss Of Function ◽  
Pupal Development ◽  
Gfp Fusion Protein ◽  
Drosophila Model ◽  
Evolutionarily Conserved ◽  
Age Dependent ◽  
Cellular Stressors ◽  
Trap Line

Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the Integrated Stress Response (ISR), an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, our data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival.

scholarly journals A protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oocyte maturation and wing development

2021 ◽  
Author(s):  
Deepika Vasudevan ◽  
Hidetaka Katow ◽  
Grace Tang ◽  
Hyung Don Ryoo
Keyword(s):  
Stress Response ◽  
Oocyte Maturation ◽  
Cellular Stress ◽  
Loss Of Function ◽  
Pupal Development ◽  
Drosophila Model ◽  
Evolutionarily Conserved ◽  
Trap Line ◽  
Adult Drosophila

Metazoans have evolved various stress response mechanisms to cope with cellular stress inflicted by external and physiological conditions. The Integrated Stress Response (ISR) is an evolutionarily conserved pathway that mediates adaptation to cellular stress via the transcription factor, ATF4. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo, and acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders photoreceptors more vulnerable to age-dependent retinal degeneration. crc mutant adult animals also show greater susceptibility to amino acid deprivation and reduced levels of known crc transcriptional targets. Furthermore, this mutant allele shows defects in wing veins and oocyte maturation, uncovering previously unknown roles for crc in the development of these tissues. Together, our data establish physiological and pathological functions of crc-mediated ISR in adult Drosophila tissues.

scholarly journals Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

2020 ◽  
Vol 295 (39) ◽  
pp. 13617-13629
Author(s):  
Clément Immarigeon ◽  
Sandra Bernat-Fabre ◽  
Emmanuelle Guillou ◽  
Alexis Verger ◽  
Elodie Prince ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Direct Interaction ◽  
Mediator Complex ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Rna Pol Ii ◽  
Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

scholarly journals Drosophila immune cells extravasate from vessels to wounds using Tre1 GPCR and Rho signaling

2018 ◽  
Vol 217 (9) ◽  
pp. 3045-3056 ◽  
Author(s):  
Leila Thuma ◽  
Deborah Carter ◽  
Helen Weavers ◽  
Paul Martin
Keyword(s):  
Immune Cells ◽  
Immune Cell ◽  
Innate Immune ◽  
Murine Models ◽  
Pupal Development ◽  
Epithelial Migration ◽  
Drosophila Model ◽  
Rate Limiting ◽  
Insight Into

Inflammation is pivotal to fight infection, clear debris, and orchestrate repair of injured tissues. Although Drosophila melanogaster have proven invaluable for studying extravascular recruitment of innate immune cells (hemocytes) to wounds, they have been somewhat neglected as viable models to investigate a key rate-limiting component of inflammation—that of immune cell extravasation across vessel walls—due to their open circulation. We have now identified a period during pupal development when wing hearts pulse hemolymph, including circulating hemocytes, through developing wing veins. Wounding near these vessels triggers local immune cell extravasation, enabling live imaging and correlative light-electron microscopy of these events in vivo. We show that RNAi knockdown of immune cell integrin blocks diapedesis, just as in vertebrates, and we uncover a novel role for Rho-like signaling through the GPCR Tre1, a gene previously implicated in the trans-epithelial migration of germ cells. We believe this new Drosophila model complements current murine models and provides new mechanistic insight into immune cell extravasation.

scholarly journals Long Term Pharmacological Perturbation of Autophagy in Mice: Are HCQ Injections a Relevant Choice?

Biomedicines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 47 ◽  
Author(s):  
Jean-Daniel Masson ◽  
Benoit Blanchet ◽  
Baptiste Periou ◽  
François-Jérôme Authier ◽  
Baharia Mograbi ◽  
...  
Keyword(s):  
Mouse Models ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Evolutionarily Conserved ◽  
Atg Genes ◽  
Conditional Inhibition ◽  
The Impact ◽  
Catabolic Process

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process whose loss-of-function has been linked to a growing list of pathologies. Knockout mouse models of key autophagy genes have been instrumental in the demonstration of the critical functions of autophagy, but they display early lethality, neurotoxicity and unwanted autophagy-independent phenotypes, limiting their applications for in vivo studies. To avoid problems encountered with autophagy-null transgenic mice, we investigated the possibility of disturbing autophagy pharmacologically in the long term. Hydroxychloroquine (HCQ) ip injections were done in juvenile and adult C57bl/6j mice, at range doses adapted from the human malaria prophylactic treatment. The impact on autophagy was assessed by western-blotting, and juvenile neurodevelopment and adult behaviours were evaluated for four months. Quite surprisingly, our results showed that HCQ treatment in conditions used in this study neither impacted autophagy in the long term in several tissues and organs nor altered neurodevelopment, adult behaviour and motor capabilities. Therefore, we recommend for future long-term in vivo studies of autophagy, to use genetic mouse models allowing conditional inhibition of selected Atg genes in appropriate lineage cells instead of HCQ treatment, until it could be successfully revisited using higher HCQ doses and/or frequencies with acceptable toxicity.

scholarly journals HSP90 is a chaperone for DLK and is required for axon injury signaling

2018 ◽  
Vol 115 (42) ◽  
pp. E9899-E9908 ◽  
Author(s):  
Scott Karney-Grobe ◽  
Alexandra Russo ◽  
Erin Frey ◽  
Jeffrey Milbrandt ◽  
Aaron DiAntonio
Keyword(s):  
Leucine Zipper ◽  
Axon Regeneration ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Loss Of Function ◽  
Hsp90 Inhibition ◽  
Axon Injury ◽  
Evolutionarily Conserved ◽  
The Stability

Peripheral nerve injury induces a robust proregenerative program that drives axon regeneration. While many regeneration-associated genes are known, the mechanisms by which injury activates them are less well-understood. To identify such mechanisms, we performed a loss-of-function pharmacological screen in cultured adult mouse sensory neurons for proteins required to activate this program. Well-characterized inhibitors were present as injury signaling was induced but were removed before axon outgrowth to identify molecules that block induction of the program. Of 480 compounds, 35 prevented injury-induced neurite regrowth. The top hits were inhibitors to heat shock protein 90 (HSP90), a chaperone with no known role in axon injury. HSP90 inhibition blocks injury-induced activation of the proregenerative transcription factor cJun and several regeneration-associated genes. These phenotypes mimic loss of the proregenerative kinase, dual leucine zipper kinase (DLK), a critical neuronal stress sensor that drives axon degeneration, axon regeneration, and cell death. HSP90 is an atypical chaperone that promotes the stability of signaling molecules. HSP90 and DLK show two hallmarks of HSP90–client relationships: (i) HSP90 binds DLK, and (ii) HSP90 inhibition leads to rapid degradation of existing DLK protein. Moreover, HSP90 is required for DLK stability in vivo, where HSP90 inhibitor reduces DLK protein in the sciatic nerve. This phenomenon is evolutionarily conserved in Drosophila. Genetic knockdown of Drosophila HSP90, Hsp83, decreases levels of Drosophila DLK, Wallenda, and blocks Wallenda-dependent synaptic terminal overgrowth and injury signaling. Our findings support the hypothesis that HSP90 chaperones DLK and is required for DLK functions, including proregenerative axon injury signaling.

scholarly journals Age-dependent emergence of neurophysiological and behavioral abnormalities in progranulin-deficient mice

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Dávid Nagy ◽  
Lauren Herl Martens ◽  
Liza Leventhal ◽  
Angela Chen ◽  
Craig Kelley ◽  
...  
Keyword(s):  
Frontotemporal Dementia ◽  
Neurodegenerative Disorder ◽  
Neuronal Loss ◽  
Loss Of Function ◽  
Behavioral Alterations ◽  
Response Latencies ◽  
Reward Seeking ◽  
Behavioral Abnormalities ◽  
Age Dependent

Abstract Background Loss-of-function mutations in the progranulin gene cause frontotemporal dementia, a genetic, heterogeneous neurodegenerative disorder. Progranulin deficiency leads to extensive neuronal loss in the frontal and temporal lobes, altered synaptic connectivity, and behavioral alterations. Methods The chronological emergence of neurophysiological and behavioral phenotypes of Grn heterozygous and homozygous mice in the dorsomedial thalamic—medial prefrontal cortical pathway were evaluated by in vivo electrophysiology and reward-seeking/processing behavior, tested between ages 3 and 12.5 months. Results Electrophysiological recordings identified a clear age-dependent deficit in the thalamocortical circuit. Both heterozygous and homozygous mice exhibited impaired input-output relationships and paired-pulse depression, but evoked response latencies were only prolonged in heterozygotes. Furthermore, we demonstrate firstly an abnormal reward-seeking/processing behavior in the homozygous mice which correlates with previously reported neuroinflammation. Conclusion Our findings indicate that murine progranulin deficiency causes age-dependent neurophysiological and behavioral abnormalities thereby indicating their validity in modeling aspects of human frontotemporal dementia.

scholarly journals Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

2020 ◽  
Vol 4 (23) ◽  
pp. 5915-5924
Author(s):  
Jana Oltova ◽  
Ondrej Svoboda ◽  
Olga Machonova ◽  
Petra Svatonova ◽  
David Traver ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Erythroid Cell ◽  
Loss Of Function ◽  
Erythroid Progenitors ◽  
Evolutionarily Conserved

Abstract Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

scholarly journals Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009794
Author(s):  
Stefano Suzzi ◽  
Reiner Ahrendt ◽  
Stefan Hans ◽  
Svetlana A. Semenova ◽  
Avinash Chekuru ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Biological Activities ◽  
Specific Cell ◽  
Loss Of Function ◽  
Developmental Abnormalities ◽  
Age Dependent ◽  
Paralog Gene ◽  
Early Developmental ◽  
The Brain

LRRK2 gain-of-function is considered a major cause of Parkinson’s disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

scholarly journals Author response: Age-dependent diastolic heart failure in an in vivo Drosophila model

2017 ◽  
Author(s):  
Matthew P Klassen ◽  
Christian J Peters ◽  
Shiwei Zhou ◽  
Hannah H Williams ◽  
Lily Yeh Jan ◽  
...  
Keyword(s):  
Heart Failure ◽  
Diastolic Heart Failure ◽  
Author Response ◽  
Drosophila Model ◽  
Age Dependent
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