The voices of Ukrainian and Russian scientists.

The brutal attack on Ukraine by the Russian Federation has shocked the world. While the world works to end the violence and help refugees, as a scientific journal, our thoughts are also with those in the scientific community who are directly or indirectly impacted by the war. We have been inspired by and applaud the labs around the world that have opened their doors to displaced scientists and remain committed to supporting scientists, whoever and wherever they are. Because science requires collaboration and trust, we urge the scientific community to continue efforts like this and to remain united, especially in times as difficult as these. In this Voices piece, we feature short comments from scientists from Ukraine and scientists from Russia. This small sampling is far from exhaustive, but our sincere thanks go to those scientists who were willing to share their thoughts on this volatile and emotionally charged situation; the views expressed are those of the contributors alone. We join the world in hoping for a swift resolution to the conflict, for the good of humanity.

The Russian invasion of Ukraine: a humanitarian tragedy and a tragedy for science.

The Invasion of Ukraine prompts us to support our Ukranian colleagues but also to keep open communication with the Russian scientists who oppose the war.

Tissue-specific regulation of translational readthrough tunes functions of the traffic jam transcription factor.

Translational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR has been identified in several eukaryotic organisms; however, its biological significance and mechanism remain unclear. Here, we quantify TR of several candidate genes in Drosophila melanogaster and characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). Using CRISPR/Cas9-generated mutant flies, we show that the TR-generated Tj isoform is expressed in a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads. Control of TR in Tj is critical for preservation of neuronal integrity and maintenance of reproductive health. The tissue-specific distribution of a release factor splice variant, eRF1H, plays a critical role in modulating differential TR of leaky stop codon contexts. Fine-tuning of gene regulatory functions of transcription factors by TR provides a potential mechanism for cell-specific regulation of gene expression.

Distant activation of Notch signaling induces stem cell niche assembly.

Here we show that multiple modes of Notch signaling activation specify the complexity of spatial cellular interactions necessary for stem cell niche assembly. In particular, we studied the formation of the germline stem cell niche in Drosophila ovaries, which is a two-step process whereby terminal filaments are formed first. Then, terminal filaments signal to the adjacent cap cell precursors, resulting in Notch signaling activation, which is necessary for the lifelong acquisition of stem cell niche cell fate. The genetic data suggest that in order to initiate the process of stem cell niche assembly, Notch signaling is activated among non-equipotent cells via distant induction, where germline Delta is delivered to somatic cells located several diameters away via cellular projections generated by primordial germ cells. At the same time, to ensure the robustness of niche formation, terminal filament cell fate can also be induced by somatic Delta via cis- or trans-inhibition. This exemplifies a double security mechanism that guarantees that the germline stem cell niche is formed, since it is indispensable for the adjacent germline precursor cells to acquire and maintain stemness necessary for successful reproduction. These findings contribute to our understanding of the formation of stem cell niches in their natural environment, which is important for stem cell biology and regenerative medicine.

Stereotypical architecture of the stem cell niche is spatiotemporally established by miR-125-dependent coordination of Notch and steroid signaling.

Stem cell niches act as signaling platforms that regulate stem cell self-renewal and sustain stem cells throughout life; however, the specific developmental events controlling their assembly are not well understood. Here, we show that during Drosophila ovarian germline stem cell niche formation, the status of Notch signaling in the cell can be reprogrammed. This is controlled via steroid-induced miR-125, which targets a negative regulator of Notch signaling, Tom. Thus, miR-125 acts as a spatiotemporal coordinator between paracrine Notch and endocrine steroid signaling. Moreover, a dual security mechanism for Notch signaling activation exists to ensure the robustness of niche assembly. Particularly, stem cell niche cells can be specified either via lateral inhibition, in which a niche cell precursor acquires Notch signal-sending status randomly, or via peripheral induction, whereby Delta is produced by a specific cell. When one mechanism is perturbed due to mutations, developmental defects or environmental stress, the remaining mechanism ensures that the niche is formed, perhaps abnormally, but still functional. This guarantees that the germline stem cells will have their residence, thereby securing progressive oogenesis and, thus, organism reproduction.

Loss of swiss cheese in Neurons Contributes to Neurodegeneration with Mitochondria Abnormalities, Reactive Oxygen Species Acceleration and Accumulation of Lipid Droplets in Drosophila Brain.

Various neurodegenerative disorders are associated with human NTE/PNPLA6 dysfunction. Mechanisms of neuropathogenesis in these diseases are far from clearly elucidated. Hereditary spastic paraplegia belongs to a type of neurodegeneration associated with NTE/PNLPLA6 and is implicated in neuron death. In this study, we used Drosophila melanogaster to investigate the consequences of neuronal knockdown of swiss cheese (sws)-the evolutionarily conserved ortholog of human NTE/PNPLA6-in vivo. Adult flies with the knockdown show longevity decline, locomotor and memory deficits, severe neurodegeneration progression in the brain, reactive oxygen species level acceleration, mitochondria abnormalities and lipid droplet accumulation. Our results suggest that SWS/NTE/PNPLA6 dysfunction in neurons induces oxidative stress and lipid metabolism alterations, involving mitochondria dynamics and lipid droplet turnover in neurodegeneration pathogenesis. We propose that there is a complex mechanism in neurological diseases such as hereditary spastic paraplegia, which includes a stress reaction, engaging mitochondria, lipid droplets and endoplasmic reticulum interplay.

miRNA targeting and alternative splicing in the stress response - events hosted by membrane-less compartments.

Stress can be temporary or chronic, and mild or acute. Depending on its extent and severity, cells either alter their metabolism, and adopt a new state, or die. Fluctuations in environmental conditions occur frequently, and such stress disturbs cellular homeostasis, but in general, stresses are reversible and last only a short time. There is increasing evidence that regulation of gene expression in response to temporal stress happens post-transcriptionally in specialized subcellular membrane-less compartments called ribonucleoprotein (RNP) granules. RNP granules assemble through a concentration-dependent liquid-liquid phase separation of RNA-binding proteins that contain low-complexity sequence domains (LCDs). Interestingly, many factors that regulate microRNA (miRNA) biogenesis and alternative splicing are RNA-binding proteins that contain LCDs and localize to stress-induced liquid-like compartments. Consequently, gene silencing through miRNAs and alternative splicing of pre-mRNAs are emerging as crucial post-transcriptional mechanisms that function on a genome-wide scale to regulate the cellular stress response. In this Review, we describe the interplay between these two post-transcriptional processes that occur in liquid-like compartments as an adaptive cellular response to stress.

Profiling of the muscle-specific dystroglycan interactome reveals the role of Hippo signaling in muscular dystrophy and age-dependent muscle atrophy.

BACKGROUND: Dystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity and caused by abnormal functioning of the ECM receptor dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be linked directly to mutations in Dg or its regulatory enzymes, implying the involvement of other, not-yet-characterized, Dg-regulating factors. To advance disease diagnostics and develop new treatment strategies, new approaches to find dystroglycanopathy-related factors should be considered. The Dg complex is highly evolutionarily conserved; therefore, model genetic organisms provide excellent systems to address this challenge. In particular, Drosophila is amenable to experiments not feasible in any other system, allowing original insights about the functional interactors of the Dg complex. METHODS: To identify new players contributing to dystroglycanopathies, we used Drosophila as a genetic muscular dystrophy model. Using mass spectrometry, we searched for muscle-specific Dg interactors. Next, in silico analyses allowed us to determine their association with diseases and pathological conditions in humans. Using immunohistochemical, biochemical, and genetic interaction approaches followed by the detailed analysis of the muscle tissue architecture, we verified Dg interaction with some of the discovered factors. Analyses of mouse muscles and myocytes were used to test if interactions are conserved in vertebrates. RESULTS: The muscle-specific Dg complexome revealed novel components that influence the efficiency of Dg function in the muscles. We identified the closest human homologs for Dg-interacting partners, determined their significant enrichment in disease-associations, and verified some of the newly identified Dg interactions. We found that Dg associates with two components of the mechanosignaling Hippo pathway: the WW domain-containing proteins Kibra and Yorkie. Importantly, this conserved interaction manages adult muscle size and integrity. CONCLUSIONS: The results presented in this study provide a new list of muscle-specific Dg interactors, further analysis of which could aid not only in the diagnosis of muscular dystrophies, but also in the development of new therapeutics. To regulate muscle fitness during aging and disease, Dg associates with Kibra and Yorkie and acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.

The histone H2B monoubiquitination regulatory pathway is required for differentiation of multipotent stem cells.

Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation.

Steroid-induced microRNA let-7 acts as a spatio-temporal code for neuronal cell fate in the developing Drosophila brain.

Mammalian neuronal stem cells produce multiple neuron types in the course of an individual's development. Similarly, neuronal progenitors in the Drosophila brain generate different types of closely related neurons that are born at specific time points during development. We found that in the post-embryonic Drosophila brain, steroid hormones act as temporal cues that specify the cell fate of mushroom body (MB) neuroblast progeny. Chronological regulation of neurogenesis is subsequently mediated by the microRNA (miRNA) let-7, absence of which causes learning impairment due to morphological MB defects. The miRNA let-7 is required to regulate the timing of α'/ß' to α/ß neuronal identity transition by targeting the transcription factor Abrupt. At a cellular level, the ecdysone-let-7-Ab signalling pathway controls the expression levels of the cell adhesion molecule Fasciclin II in developing neurons that ultimately influences their differentiation. Our data propose a novel role for miRNAs as transducers between chronologically regulated developmental signalling and physical cell adhesion.

TfAP-2 is required for night sleep in Drosophila.

BACKGROUND: The AP-2 transcription factor APTF-1 is crucially required for developmentally controlled sleep behavior in Caenorhabditis elegans larvae. Its human ortholog, TFAP-2beta, causes Char disease and has also been linked to sleep disorders. These data suggest that AP-2 transcription factors may be highly conserved regulators of various types of sleep behavior. Here, we tested the idea that AP-2 controls adult sleep in Drosophila. RESULTS: Drosophila has one AP-2 ortholog called TfAP-2, which is essential for viability. To investigate its potential role in sleep behavior and neural development, we specifically downregulated TfAP-2 in the nervous system. We found that neuronal TfAP-2 knockdown almost completely abolished night sleep but did not affect day sleep. TfAP-2 insufficiency affected nervous system development. Conditional TfAP-2 knockdown in the adult also produced a modest sleep phenotype, suggesting that TfAP-2 acts both in larval as well as in differentiated neurons. CONCLUSIONS: Thus, our results show that AP-2 transcription factors are highly conserved regulators of development and sleep.

Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation.

Previously, it has been shown that in Drosophila steroid hormones are required for progression of oogenesis during late stages of egg maturation. Here, we show that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay to switch on a differentiation programme. This differentiation impediment is associated with reduced TGF-ß signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed.

Unraveling the link between neuropathy target esterase NTE/SWS, lysosomal storage diseases, inflammation, abnormal fatty acid metabolism, and leaky brain barrier.

Mutations in Drosophila Swiss cheese (SWS) gene or its vertebrate orthologue neuropathy target esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.

Drosophila MIC10b can polymerize into cristae-shaping filaments.

Cristae are invaginations of the mitochondrial inner membrane that are crucial for cellular energy metabolism. The formation of cristae requires the presence of a protein complex known as MICOS, which is conserved across eukaryotic species. One of the subunits of this complex, MIC10, is a transmembrane protein that supports cristae formation by oligomerization. In Drosophila melanogaster, three MIC10-like proteins with different tissue-specific expression patterns exist. We demonstrate that CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Its loss destabilizes MICOS, disturbs cristae architecture, and reduces the life span and fertility of flies. We show that DmMIC10b has a unique ability to polymerize into bundles of filaments, which can remodel mitochondrial crista membranes. The formation of these filaments relies on conserved glycine and cysteine residues, and can be suppressed by the co-expression of other Drosophila MICOS proteins. These findings provide new insights into the regulation of MICOS in flies, and suggest potential mechanisms for the maintenance of mitochondrial ultrastructure.

Signaling through the dystrophin glycoprotein complex affects the stress-dependent transcriptome in Drosophila.

Deficiencies in the human dystrophin glycoprotein complex (DGC), which links the extracellular matrix with the intracellular cytoskeleton, cause muscular dystrophies, a group of incurable disorders associated with heterogeneous muscle, brain and eye abnormalities. Stresses such as nutrient deprivation and aging cause muscle wasting, which can be exacerbated by reduced levels of the DGC in membranes, the integrity of which is vital for muscle health and function. Moreover, the DGC operates in multiple signaling pathways, demonstrating an important function in gene expression regulation. To advance disease diagnostics and treatment strategies, we strive to understand the genetic pathways that are perturbed by DGC mutations. Here, we utilized a Drosophila model to investigate the transcriptomic changes in mutants of four DGC components under temperature and metabolic stress. We identified DGC-dependent genes, stress-dependent genes and genes dependent on the DGC for a proper stress response, confirming a novel function of the DGC in stress-response signaling. This perspective yields new insights into the etiology of muscular dystrophy symptoms, possible treatment directions and a better understanding of DGC signaling and regulation under normal and stress conditions.

Stress and muscular dystrophy: a genetic screen for dystroglycan and dystrophin interactors in Drosophila identifies cellular stress response components.

In Drosophila, like in humans, Dystrophin Glycoprotein Complex (DGC) deficiencies cause a life span shortening disease, associated with muscle dysfunction. We performed the first in vivo genetic interaction screen in ageing dystrophic muscles and identified genes that have not been shown before to have a role in the development of muscular dystrophy and interact with dystrophin and/or dystroglycan. Mutations in many of the found interacting genes cause age-dependent morphological and heat-induced physiological defects in muscles, suggesting their importance in the tissue. Majority of them is phylogenetically conserved and implicated in human disorders, mainly tumors and myopathies. Functionally they can be divided into three main categories: proteins involved in communication between muscle and neuron, and interestingly, in mechanical and cellular stress response pathways. Our data show that stress induces muscle degeneration and accelerates age-dependent muscular dystrophy. Dystrophic muscles are already compromised; and as a consequence they are less adaptive and more sensitive to energetic stress and to changes in the ambient temperature. However, only dystroglycan, but not dystrophin deficiency causes extreme myodegeneration induced by energetic stress suggesting that dystroglycan might be a component of the low-energy pathway and act as a transducer of energetic stress in normal and dystrophic muscles.

Dg-Dys-Syn1 signaling in Drosophila regulates the microRNA profile.

BACKGROUND: The Dystrophin Glycoprotein Complex (DGC) is at the center of significant inheritable diseases, such as muscular dystrophies that can be fatal and impair neuronal function in addition to muscle degeneration. Recent evidence has shown that it can control cellular homeostasis and work via Dystrophin signaling to regulate microRNA gene expression which implies that disease phenotypes hide an entourage of regulatory and homeostatic anomalies. Uncovering these hidden processes could shed new light on the importance of proper DGC function for an organism's overall welfare and bring forth new ideas for treatments. RESULTS: To better understand a role for the DGC in these processes, we used the genetically advantageous Drosophila muscular dystrophy model to conduct a whole animal microarray screen. Since we have recently found that dystrophic symptoms can be caused by stress even in wild type animals and are enhanced in mutants, we screened stressed animals for microRNA misregulation as well. We were able to define microRNAs misregulated due to stress and/or dystrophy. Our results support the hypothesis that there is a Dystrophin and Dystroglycan dependent circuitry of processes linking stress response, dystrophic conditions and cellular signaling and that microRNAs play an important role in this network. Verification of a subset of our results was conducted via q-PCR and revealed that miR-956, miR-980 and miR-252 are regulated via a Dystroglycan-Dystrophin-Syntrophin dependent pathway. CONCLUSIONS: The results presented in this study support the hypothesis that there is a Dystrophin and Dystroglycan dependent circuitry of processes that includes regulation of microRNAs. Dystrophin signaling has already been found to occur in mammalian musculature; however, our data reveals that this regulation is evolutionarily conserved and also present in at least neuronal tissues. Our data imply that Dystroglycan-Dystrophin-Syntrophin signaling through control of multiple microRNAs is involved in highly managed regulation of gene expression required to adapt cellular homeostasis that is compromised under stress and dystrophic conditions.

Notch signaling sculpts the stem cell niche.

Adult stem cells depend on their niches for regulatory signaling that controls their maintenance, division, and their progeny differentiation. While communication between various types of stem cells and their niches is becoming clearer, the process of stem cell niche establishment is still not very well understood. Model genetic organisms provide simplified systems to address various complex questions, for example, how is a stem cell niche formed? What signaling cascades induce the stem cell niche formation? Are the mechanisms of stem cell niche formation conserved? Notch signaling is an evolutionarily conserved pathway first identified in fruit flies, crucial in fate acquisition and spatiotemporal patterning. While the core logic behind its activity is fairly simple and requires direct cell-cell interaction, it reaches an astonishing complexity and versatility by combining its different modes of action. Subtleties such as equivalency between communicating cells, their physical distance, receptor and ligand processing, and endocytosis can have an effect on the way the events unfold, and this review explores some important general mechanisms of action, later on focusing on its involvement in stem cell niche formation. First, looking at invertebrates, we will examine how Notch signaling induces the formation of germline stem cell niche in male and female Drosophila. In the developing testis, a group of somatic gonadal precursor cells receive Delta signals from the gut, activating Notch signaling and sealing their fate as niche cells even before larval hatching. Meanwhile, the ovarian germline stem cell niche is built later during late larval stages and requires a two-step process that involves terminal filament formation and cap cell specification. Intriguingly, double security mechanisms of Notch signaling activation coordinated by the soma or the germline control both steps to ensure the robustness of niche assembly. Second, in the vast domains of mammalian cellular signaling, there is an emerging picture of Notch being an active player in a variety of tissues in health and disease. Notch involvement has been shown in stem cell niche establishment in multiple organs, including the brain, muscle, and intestine, where the stem cell niches are essential for the maintenance of adult stem cells. But adult stem cells are not the only cells looking for a home. Cancer stem cells use Notch signaling at specific stages to gain an advantage over endogenous tissue and overpower it, at the same time acquiring migratory and invasive abilities to claim new tissues (e.g., bone) as their territory. Moreover, in vitro models such as organoids reveal similar Notch employment when it comes to the developing stem cell niches. Therefore, a better understanding of the processes regulating stem cell niche assembly is key for the fields of stem cell biology and regenerative medicines.

New dystrophin/dystroglycan interactors control neuron behavior in Drosophila eye.

BACKGROUND: The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Previously we performed an array of genetic screens using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, we have here continued to analyze the DGC modifiers' function in the developing Drosophila brain and eye. RESULTS: Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, we have determined the function of previously screened components and their genetic interaction with the DGC in this tissue. Our study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. We determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. CONCLUSIONS: Our data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton.

Exocyst-mediated membrane trafficking of the lissencephaly-associated ECM receptor dystroglycan is required for proper brain compartmentalization.

To assemble a brain, differentiating neurons must make proper connections and establish specialized brain compartments. Abnormal levels of cell adhesion molecules disrupt these processes. Dystroglycan (Dg) is a major non-integrin cell adhesion receptor, deregulation of which is associated with dramatic neuroanatomical defects such as lissencephaly type II or cobblestone brain. The previously established Drosophila model for cobblestone lissencephaly was used to understand how Dg is regulated in the brain. During development, Dg has a spatiotemporally dynamic expression pattern, fine-tuning of which is crucial for accurate brain assembly. In addition, mass spectrometry analyses identified numerous components associated with Dg in neurons, including several proteins of the exocyst complex. Data show that exocyst-based membrane trafficking of Dg allows its distinct expression pattern, essential for proper brain morphogenesis. Further studies of the Dg neuronal interactome will allow identification of new factors involved in the development of dystroglycanopathies and advance disease diagnostics in humans.