Notch Signaling: An Emerging Paradigm in the Pathogenesis of Reproductive Disorders and Diverse Pathological Conditions.

The highly conserved Notch pathway, a pillar of juxtacrine signaling, orchestrates intricate intercellular communication, governing diverse developmental and homeostatic processes through a tightly regulated cascade of proteolytic cleavages. This pathway, culminating in the migration of the Notch intracellular domain (NICD) to the nucleus and the subsequent activation of downstream target genes, exerts a profound influence on a plethora of molecular processes, including cell cycle progression, lineage specification, cell-cell adhesion, and fate determination. Accumulating evidence underscores the pivotal role of Notch dysregulation, encompassing both gain and loss-of-function mutations, in the pathogenesis of numerous human diseases. This review delves deep into the multifaceted roles of Notch signaling in cellular dynamics, encompassing proliferation, differentiation, polarity maintenance, epithelial-mesenchymal transition (EMT), tissue regeneration/remodeling, and its intricate interplay with other signaling pathways. We then focus on the emerging landscape of Notch aberrations in gynecological pathologies predisposing individuals to infertility. By highlighting the exquisite conservation of Notch signaling in Drosophila and its power as a model organism, we pave the way for further dissection of disease mechanisms and potential therapeutic interventions through targeted modulation of this master regulatory pathway.

Dual-Color Live Imaging of Adult Muscle Stem Cells in the Embryonic Tissues of Drosophila melanogaster.

Adult muscle stem cells (MuSCs) show remarkable capability in repairing injured tissues. Studying MuSCs in suitable model organisms, which show strong homology with vertebrate counterparts, helps in dissecting the mechanisms regulating their behavior. Additionally, ease of handling and access to technological tools make model organisms well suited for studying biological processes that are conserved across species. MuSCs quiescence, proliferation, and migration are regulated by various input of signals from the surrounding tissues that constitute the MuSCs niche. Observing MuSCs along with their niche in vivo through live imaging provides key information on how MuSCs behave in quiescent and activated states. Drosophila melanogaster is well known for its genetic tool arsenal and the similarity of its different biological processes with vertebrates. Hence, it is widely used to study different types of stem cells. Gained knowledge could then be extrapolated to the vertebrate/mammalian homologous systems to enhance our knowledge in stem cell fields. In this protocol, we discuss how to perform live cell imaging of Drosophila MuSCs, called adult muscle precursors (AMPs) at embryonic stages, using dual-color labelling to visualize both AMPs and the surrounding tissues. This dual-color fluorescent labelling enables the observation of in vivo behavior of two types of cells simultaneously and provides key information on their interactions. The originality of this protocol resides in its biological application to MuSCs and their niche.

Glycosylation in SARS-CoV-2 variants: A path to infection and recovery.

Glycan is an essential molecule that controls and drives life in a precise direction. The paucity of research in glycobiology may impede the significance of its role in the pandemic guidelines. The SARS-CoV-2 spike protein is heavily glycosylated, with 22 putative N-glycosylation sites and 17 potential O-glycosylation sites discovered thus far. It is the anchor point to the host cell ACE2 receptor, TMPRSS2, and many other host proteins that can be recognized by their immune system; hence, glycosylation is considered the primary target of vaccine development. Therefore, it is essential to know how this surface glycan plays a role in viral entry, infection, transmission, antigen, antibody responses, and disease progression. Although the vaccines are developed and applied against COVID-19, the proficiency of the immunizations is not accomplished with the current mutant variations. The role of glycosylation in SARS-CoV-2 and its receptor ACE2 with respect to other putative cell glycan receptors and the significance of glycan in host cell immunity in COVID-19 are discussed in this paper. Hence, the molecular signature of the glycan in the coronavirus infection can be incorporated into the mainstream therapeutic process.

Emerging roles of metazoan cell cycle regulators as coordinators of the cell cycle and differentiation.

In multicellular organisms, cell proliferation must be tightly coordinated with other developmental processes to form functional tissues and organs. Despite significant advances in our understanding of how the cell cycle is controlled by conserved cell-cycle regulators (CCRs), how the cell cycle is coordinated with cell differentiation in metazoan organisms and how CCRs contribute to this process remain poorly understood. Here, we review the emerging roles of metazoan CCRs as intracellular proliferation-differentiation coordinators in multicellular organisms. We illustrate how major CCRs regulate cellular events that are required for cell fate acquisition and subsequent differentiation. To this end, CCRs employ diverse mechanisms, some of which are separable from those underpinning the conventional cell-cycle-regulatory functions of CCRs. By controlling cell-type-specific specification/differentiation processes alongside the progression of the cell cycle, CCRs enable spatiotemporal coupling between differentiation and cell proliferation in various developmental contexts in vivo. We discuss the significance and implications of this underappreciated role of metazoan CCRs for development, disease and evolution.

Insulin-dependent Non-canonical Activation of Notch in Drosophila: A Story of Notch-Induced Muscle Stem Cell Proliferation.

Notch plays multiple roles both in development and in adult tissue homeostasis. Notch was first identified in Drosophila in which it has then been extensively studied. Among the flag-ship Notch functions we could mention its capacity to keep precursor and stem cells in a nondifferentiated state but also its ability to activate cell proliferation that in some contexts could led to cancer. In general, both these functions involve, canonical, ligand-dependent Notch activation. However, a ligand-independent Notch activation has also been described in a few cellular contexts. Here, we focus on one of such contexts, Drosophila muscle stem cells, called AMPs, and discuss how insulin-dependent noncanonical activation of Notch pushes quiescent AMPs to proliferation.

miRNA suppression of a Notch repressor directs non-neuronal fate in Drosophila mechanosensory organs.

Although there is abundant evidence that individual microRNA (miRNA) loci repress large cohorts of targets, large-scale knockout studies suggest that most miRNAs are phenotypically dispensable. Here, we identify a rare case of developmental cell specification that is highly dependent on miRNA control of an individual target. We observe that binary cell fate choice in the Drosophila melanogaster peripheral sensory organ lineage is controlled by the non-neuronally expressed mir-279/996 cluster, with a majority of notum sensory organs exhibiting transformation of sheath cells into ectopic neurons. The mir-279/996 defect phenocopies Notch loss of function during the sheath-neuron cell fate decision, suggesting the miRNAs facilitate Notch signaling. Consistent with this, mir-279/996 knockouts are strongly enhanced by Notch heterozygosity, and activated nuclear Notch is impaired in the miRNA mutant. Although Hairless (H) is the canonical nuclear Notch pathway inhibitor, and H heterozygotes exhibit bristle cell fate phenotypes reflecting gain-of-Notch signaling, H/+ does not rescue mir-279/996 mutants. Instead, we identify Insensible (Insb), another neural nuclear Notch pathway inhibitor, as a critical direct miR-279/996 target. Insb is posttranscriptionally restricted to neurons by these miRNAs, and its heterozygosity strongly suppresses ectopic peripheral nervous system neurons in mir-279/996 mutants. Thus, proper assembly of multicellular mechanosensory organs requires a double-negative circuit involving miRNA-mediated suppression of a Notch repressor to assign non-neuronal cell fate.

New genes often acquire male-specific functions but rarely become essential in Drosophila.

Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of "conserved, essential" and "young, RNAi-lethal" genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function.

Muscle niche-driven Insulin-Notch-Myc cascade reactivates dormant Adult Muscle Precursors in Drosophila.

How stem cells specified during development keep their non-differentiated quiescent state, and how they are reactivated, remain poorly understood. Here, we applied a Drosophila model to follow in vivo behavior of adult muscle precursors (AMPs), the transient fruit fly muscle stem cells. We report that emerging AMPs send out thin filopodia that make contact with neighboring muscles. AMPs keep their filopodia-based association with muscles throughout their dormant state but also when they start to proliferate, suggesting that muscles could play a role in AMP reactivation. Indeed, our genetic analyses indicate that muscles send inductive dIlp6 signals that switch the Insulin pathway ON in closely associated AMPs. This leads to the activation of Notch, which regulates AMP proliferation via dMyc. Altogether, we report that Drosophila AMPs display homing behavior to muscle niche and that the niche-driven Insulin-Notch-dMyc cascade plays a key role in setting the activated state of AMPs.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos.

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

Specification and behavior of AMPs, muscle-committed transient Drosophila stem cells.

During development, transient stem cells play critical roles in the formation of specific tissues. Adult Muscle Precursors (AMPs) are at the origin of all adult Drosophila muscles and as we report here represent a novel population of muscle-committed transient stem cells. Similar to vertebrate muscle stem cells, AMPs keep Notch signaling active and express Enhancer of split m6 (E(spl)m6) gene, a read-out of Notch pathway. To get insights into AMP cell specification we performed a gain-of-function screen and found that the rhomboid-triggered Epidermal Growth Factor (EGF) signaling pathway controls both the specification and the subsequent maintenance of AMPs. Our findings are supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Interestingly, by targeting GFP to the AMP cell membranes we also demonstrated that AMPs send long cellular processes and form a network of interconnected cells. As revealed by laser ablation experiments, the main role of AMP cell connections is to maintain their correct spatial positioning.

Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway.

In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as we demonstrate here, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, we show that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, we performed a gain-of-function screen with a special focus on lateral AMPs expressing the homeobox gene ladybird. Our data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, our results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance.

Macromolecular uptake in Drosophila pericardial cells requires rudhira function.

The vertebrate reticuloendothelial system (RES) functions to remove potentially damaging macromolecules, such as excess hormones, immune-peptides and -complexes, bacterial-endotoxins, microorganisms and tumor cells. Insect hemocytes and nephrocytes - which include pericardial cells (PCs) and garland cells - are thought to be functionally equivalent to the RES. Although the ability of both vertebrate scavenger endothelial cells (SECs) and PCs to sequester colloidal and soluble macromolecules has been demonstrated the molecular mechanism of this function remains to be investigated. We report here the functional characterization of Drosophila larval PCs with important insights into their cellular uptake pathways. We demonstrate the nephrocyte function of PCs in live animals. We also develop and use live-cell assays to show that PCs take up soluble macromolecules in a Dynamin-dependent manner and colloids by a Dynamin-independent pathway. We had earlier identified Drosophila rudhira (Drudh) as a specific marker for PCs. Using RNAi mediated knock-down we show that Drudh regulates macropinocytic uptake in PCs. Our study establishes important functions for Drosophila PCs, describes methods to identify and study them, provides a genetic handle for further investigation of their role in maintaining homeostasis and demonstrates that they perform key subsets of the roles played by the vertebrate RES.

Gene expression analysis in post-embryonic pericardial cells of Drosophila.

Increasing evidence suggests conservation of cardiovascular molecules between vertebrates and invertebrates. Vertebrate Rudhira, an evolutionary conserved WD40 protein is expressed during primitive erythropoiesis, neoangiogenesis and tumors. We report here the expression profile of the Drosophila ortholog of Rudhira (DRudh) in the fly life cycle. DRudh is expressed specifically in all post-embryonic pericardial cells (PCs) and garland cells (GCs). This is the first report of a cytoplasmic marker highly specific to post-embryonic PCs. Embryonic PCs belong to three distinct genetic classes based on Odd-skipped (Odd), Even-skipped (Eve) and Tinman (Tin) expression. To identify which among these three classes of PCs expresses DRudh in post-embryonic stages, we analyzed expression of embryonic PC markers in the post-embryonic stages. Unlike in the embryo all larval PCs show an identical gene expression profile. While Odd and Eve expression is mutually exclusive in the embryonic PCs, these two markers are co-expressed in larval PCs but show a distinct subcellular localization. Tin is not expressed in any post-embryonic PC. Additionally larval PCs also express the GATA factor, Serpent (Srp) and the extracellular matrix protein, Pericardin (Prc). While PC number is known to decrease post-embryogenesis, which of the Odd or Eve lineage embryonic PCs persists is not known. Co-expression of the two distinct lineage markers only in post-embryonic stages indicates a complex temporal regulation of gene expression in PCs.

Post-embryonic pericardial cells of Drosophila are required for overcoming toxic stress but not for cardiac function or adult development.

The Drosophila heart is composed of two cell types: cardioblasts (CB) and pericardial cells (PC). Whereas CBs act to maintain rhythmic contractions, the functions of accessory PCs are not clear. The close association between these two cell types has led to speculation of a cardio-regulatory role for PCs. However, we find that viability and cardiac function are normal in larvae following post-embryonic ablation of PCs by induced cell death. Removal of PCs during the larval instars or before metamorphosis results in viable and fertile adults. Interestingly, such animals have a reduced lifespan and increased sensitivity to toxic chemicals. Thus, although PCs may have an embryonic role in cardiogenesis, they do not appear to play a part later in cardiac function as suggested. However, the role of PCs in the uptake and sequestering of toxins, their sensitivity to toxic stress and the decreased lifespan of animals without PCs indicate the importance of PCs in organismal homeostasis.