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The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process. [Display omitted] • Enhanced Mef2 expression in wing disc myoblasts formed striated structures, with no evidence of extensive myoblast fusion. • There was heterogeneity in MEF2 accumulation, and regional variation in muscle differentiation. • Most up-regulated genes were associated with myofibril assembly. • MEF2 primarily activated the larval and not the adult myogenic program. [ABSTRACT FROM AUTHOR]

The larval stage of the Drosophila melanogaster life cycle is characterized by rapid growth and nutrient storage that occur over three instar stages separated by molts. In the third instar, the steroid hormone ecdysone drives key developmental processes and behaviors that occur in a temporally-controlled sequence and prepare the animal to undergo metamorphosis. Accurately staging Drosophila larvae within the final third instar is critical due to the rapid developmental progress at this stage, but it is challenging because the rate of development varies widely across a population of animals even if eggs are laid within a short period of time. Moreover, many methods to stage third instar larvae are cumbersome, and inherent variability in the rate of development confounds some of these approaches. Here we demonstrate the usefulness of the Sgs3-GFP transgene, a fusion of the Salivary gland secretion 3 (Sgs3) and GFP proteins, for staging third instar larvae. Sgs3-GFP is expressed in the salivary glands in an ecdysone-dependent manner from the midpoint of the third instar, and its expression pattern changes reproducibly as larvae progress through the third instar. We show that Sgs3-GFP can easily be incorporated into experiments, that it allows collection of developmentally-equivalent individuals from a mixed population of larvae, and that its use enables precise assessment of changing levels of hormones, metabolites, and gene expression during the second half of the third instar. [Display omitted] • Use of inaccurately-staged Drosophila third instar larvae introduces experimental error. • Inaccurate staging can impact assessment and discovery of mutant phenotypes. • Sgs3-GFP facilitates collection of developmentally-equivalent third instar larvae. • Sgs3-GFP is easy to use and allows fast visual staging of larvae. • Staging third instar larvae with Sgs3-GFP increases accuracy and reliability of data. [ABSTRACT FROM AUTHOR]

Drosophila melanogaster is an ideal model organism for investigating spermatogenesis due to its powerful genetics, conserved genes and visible morphology of germ cells during sperm production. Our previous work revealed that ocnus (ocn) knockdown resulted in male sterility, and CG9920 was identified as a significantly downregulated protein in fly abdomen after ocn knockdown, suggesting a role of CG9920 in male reproduction. In this study, we found that CG9920 was highly expressed in fly testes. CG9920 knockdown in fly testes caused male infertility with no mature sperms in seminal vesicles. Immunofluorescence staining showed that depletion of CG9920 resulted in scattered spermatid nuclear bundles, fewer elongation cones that did not migrate to the anterior region of the testis, and almost no individualization complexes. Transmission electron microscopy revealed that CG9920 knockdown severely disrupted mitochondrial morphogenesis during spermatogenesis. Notably, we found that CG9920 might not directly interact with Ocn, but rather was inhibited by STAT92E, which itself was indirectly affected by Ocn. We propose a possible novel pathway essential for spermatogenesis in D. melanogaster , whereby Ocn indirectly induces CG9920 expression, potentially counteracting its inhibition by the JAK-STAT signaling pathway. [Display omitted] • CG9920 knockdown in germline caused male sterility with no mature sperms. • Depletion of CG9920 resulted in scattered spermatid nuclear bundles. • CG9920 knockdown led to fewer elongation cones and almost no ICs. • CG9920 knockdown disrupted mitochondrial morphogenesis during spermatogenesis. • CG9920 might be regulated by Ocnus through the JAK-STAT pathway. [ABSTRACT FROM AUTHOR]

Steroid hormones play various physiological roles including metabolism and reproduction. Steroid hormones in insects are ecdysteroids, and the major form in Drosophila melanogaster is ecdysone. In Drosophila males, the accessory gland is responsive to nutrient-dependent regulation of fertility/fecundity. The accessory gland is composed of two types of binucleated epithelial cells: a main cell and a secondary cell (SC). The transcription factors Defective proventriculus (Dve), Abdominal-B, and Ecdysone receptors (EcRs) are strongly expressed in adult SCs. We show that this EcR expression is regulated by parallel pathways of nutrient signaling and the Dve activity. Induction of Dve expression is also dependent on nutrient signaling, and it becomes nutrient signal-independent during a restricted period of development. Forced dve expression during the restricted period significantly increased the number of SCs. Here, we provide evidence that the level of nutrient signal-dependent Dve expression during the restricted period determines the number of SCs, and that ecdysone signaling is also crucial to optimize male fecundity through nutrient signal-dependent survival and maturation of SCs. [Display omitted] • Expression levels of Dve and EcR are nutrient signal-dependent. • Nutrient conditions are sensed in a restricted period (critical period). • Dve expression changes to a nutrient-signal independent mode. • The expression level of Dve determines the number of SCs. [ABSTRACT FROM AUTHOR]

Developmental time is a fundamental life history trait that affects the reproductive success of animals. Developmental time is known to be regulated by many genes and environmental conditions, yet mechanistic understandings of how various cellular processes influence the developmental timing of an organism are lacking. The nervous system is known to control key processes that affect developmental time, including the release of hormones that signal transitions between developmental stages. Here we show that the excitability of neurons plays a crucial role in modulating developmental time. Genetic manipulation of neuronal excitability in Drosophila melanogaster alters developmental time, which is faster in animals with increased neuronal excitability. We find that selectively modulating the excitability of peptidergic neurons is sufficient to alter developmental time, suggesting the intriguing hypothesis that the impact of neuronal excitability on DT may be at least partially mediated by peptidergic regulation of hormone release. This effect of neuronal excitability on developmental time is seen during embryogenesis and later developmental stages. Observed phenotypic plasticity in the effect of genetically increasing neuronal excitability at different temperatures, a condition also known to modulate excitability, suggests there is an optimal level of neuronal excitability, in terms of shortening DT. Together, our data highlight a novel connection between neuronal excitability and developmental time, with broad implications related to organismal physiology and evolution. • Increasing neuronal excitability shortens developmental time. • Peptidergic neuron excitability regulates developmental time. • Genes and environments interact to affect development. [ABSTRACT FROM AUTHOR]

Notch signaling controls numerous key cellular processes including cell fate determination and cell proliferation. Its malfunction has been linked to many developmental abnormalities and human disorders. Overactivation of Notch signaling is shown to be oncogenic. Retention of excess Notch protein in the endoplasmic reticulum (ER) can lead to altered Notch signaling and cell fate, but the mechanism is not well understood. In this study, we show that V5-tagged or untagged exogenous Notch is retained in the ER when overexpressed in fly tissues. Furthermore, we show that Notch retention in the ER leads to robust ER enlargement and elicits a rough eye phenotype. Gain-of-function of unfolded protein response (UPR) factors IRE1 or spliced Xbp1 (Xbp1-s) alleviates Notch accumulation in the ER, restores ER morphology and ameliorates the rough eye phenotype. Our results uncover a pivotal role of the IRE1/Xbp1 axis in regulating the detrimental effect of ER-localized excess Notch protein during development and tissue homeostasis. Schematic representation of Notch protein distribution in the ER. When IRE1 activity is low and ER overloaded with excess Notch protein, the ER membrane is remodeled to form enlarged structures. On the other hand, high IRE1 activity would lead to the clearance of ER-localized excess Notch protein and maintenance of ER homeostasis. EGF repeats: epidermal growth factor (EGF) repeats, NRR: negative regulatory region, NICD: Notch intracellular domain. [Display omitted] • Excess Notch causes developmental defects in Drosophila. • Exogenous Notch proteins accumulate in the ER. • The level of excess Notch in the ER is modulated by the IRE1/Xbp1 axis. • Gain-of-function of IRE1 or Xbp1-s ameliorates the rough eye phenotype caused by excess Notch. [ABSTRACT FROM AUTHOR]

The self-organization of cells during development is essential for the formation of healthy tissues and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. We investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. We found that Myosin XV is required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. We found that Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV isoforms are present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms. [Display omitted] • Loss of MyoXV leads to more and longer signaling filopodia during bristle patterning. • MyoXV dependent disruption of signaling filopodia leads to decreased Notch signaling. • The motor activity of MyoXV regulates signaling filopodia formation. [ABSTRACT FROM AUTHOR]

Many organs contain adult stem cells (ASCs) to replace cells due to damage, disease, or normal tissue turnover. ASCs can divide asymmetrically, giving rise to a new copy of themselves (self-renewal) and a sister that commits to a specific cell type (differentiation). Decades of research have led to the identification of pleiotropic genes whose loss or gain of function affect diverse aspects of normal ASC biology. Genome-wide screens of these so-called genetic "master regulator" (MR) genes, have pointed to hundreds of putative targets that could serve as their downstream effectors. Here, we experimentally validate and characterize the regulation of several putative targets of Escargot (Esg) and the Signal Transducer and Activator of Transcription (Stat92E, a.k.a. STAT), two known MRs in Drosophila intestinal stem cells (ISCs). Our results indicate that regardless of bioinformatic predictions, most experimentally validated targets show a profile of gene expression that is consistent with co-regulation by both Esg and STAT, fitting a rather limited set of co-regulatory modalities. A bioinformatic analysis of proximal regulatory sequences in specific subsets of co-regulated targets identified additional transcription factors that might cooperate with Esg and STAT in modulating their transcription. Lastly, in vivo manipulations of validated targets rarely phenocopied the effects of manipulating Esg and STAT, suggesting the existence of complex genetic interactions among downstream targets of these two MR genes during ISC homeostasis. [Display omitted] • DNA binding is a strong predictor of regulation by stem cell master regulator genes. • Master regulators share targets more often than predicted bioinformatically. • Shared targets conform to a limited set of co-regulatory modalities. • Manipulating targets rarely phenocopies the manipulation of their master regulators. [ABSTRACT FROM AUTHOR]

Characterization of gene regulatory networks is fundamental to understanding homeostatic development. This process can be simplified by analyzing relatively simple genomes such as the genome of Drosophila melanogaster. In this work we have developed a computational framework in Drosophila to explore for the presence of gene regulatory circuits between two large groups of transcriptional regulators: the epigenetic group of the Polycomb/trithorax (PcG/trxG) proteins and the microRNAs (miRNAs). We have searched genome-wide for miRNA targets in PcG / trxG transcripts as well as for Polycomb Response Elements (PREs) in miRNA genes. Our results show that 10% of the analyzed miRNAs could be controlling PcG/trxG gene expression, while 40% of those miRNAs are putatively controlled by the selected set of PcG/trxG proteins. The integration of these analyses has resulted in the predicted existence of 3 classes of miRNA-PcG/trxG crosstalk interactions that define potential regulatory circuits. In the first class, miRNA-PcG circuits are defined by miRNAs that reciprocally crosstalk with PcG. In the second, miRNA-trxG circuits are defined by miRNAs that reciprocally crosstalk with trxG. In the third class, miRNA-PcG/trxG shared circuits are defined by miRNAs that crosstalk with both PcG and trxG regulators. These putative regulatory circuits may uncover a novel mechanism in Drosophila for the control of PcG/trxG and miRNAs levels of expression. The computational framework developed here for Drosophila melanogaster can serve as a model case for similar analyses in other species. Moreover, our work provides, for the first time, a new and useful resource for the Drosophila community to consult prior to experimental studies investigating the epigenetic regulatory networks of miRNA-PcG/trxG mediated gene expression. [Display omitted] • A computational framework to define epigenetic regulatory circuits in Drosophila. • Regulatory interactions between microRNAs and Polycomb (PcG)/ trithorax (trxG) genes. • 35 and 15 microRNAs potentially regulate PcG and trxG gene expression respectively. • 106 pre-miRNAs are potentially regulated by PcG/trxG proteins. • miRNA-PcG/trxG crosstalk interactions define 21 gene regulatory circuits. [ABSTRACT FROM AUTHOR]

Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3 μm diameter) alternating with thin segments (>0.1 μm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.

Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. We found previously that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, we named the gene anion exchanger 2 (ae2). Here, we generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, we find that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, we find that the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and our studies provide new genetic tools to investigate the mechanisms by which this occurs.

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.

During brain development of Drosophila melanogaster many transcription factors are involved in regulating neural fate and morphogenesis. In our study we show that the transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, plays an important role in this process. Otp is expressed in a stable pattern in defined lineages from mid-embryonic stages into the adult brain and therefore a very stable marker for these lineages. We determined the abundance of the two different otp transcripts in the brain and hindgut during development using qPCR. CRISPR/Cas9 generated otp mutants of the longer protein form significantly affect the expression of Otp in specific areas. We generated an otp enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the complete expression of otp during development except the embryonic hindgut expression. Since in the embryo, the expression of Otp is posttranscriptionally regulated, we looked for putative miRNAs interacting with the otp 3′UTR, and identified microRNA-252 as a candidate. Further analyses with mutated and deleted forms of the microRNA-252 interacting sequence in the otp 3′UTR demonstrate an in vivo interaction of microRNA-252 with the otp 3′UTR. An effect of this interaction is seen in the adult brain, where Otp expression is partially abolished in a knockout strain of microRNA-252. Our results show that Otp is another important factor for brain development in Drosophila melanogaster. [Display omitted] • Otp expression in defined lineages from mid-embryonic stages into the adult brain. • Quantification of different otp transcript forms using qPCR. • Generation of an otp enhancer trap strain mimicing otp expression during development. • Regulation of otp by microRNA-252. [ABSTRACT FROM AUTHOR]

Since empty-spiracles (ems) was identified and characterized in Drosophila melanogaster as a head-gap gene, several studies have been carried out in other insect orders to confirm its evolutionary conserved function. Using the blood-sucking bug Rhodnius prolixus as biological model, we found an ems transcript with three highly conserved regions: Box-A, Box-B, and the homeodomain. R. prolixus embryos silenced by parental RNAi for two of these ems conserved regions showed both maternal and zygotic defects. Rp-emsB fragment results in early lethal embryogenesis, with eggs without any embryonic structure inside. Rp-emsB expression pattern is only maternally expressed and localized in the ovary tropharium, follicular cells, and in the unfertilized female pronucleus. Rp-emsA fragment is zygotically expressed during early blastoderm formation until late developmental stages in two main patterns: anterior in the antennal segment, and in a segmentary in the neuroblast and tracheal pits. R. prolixus knockdown embryos for Rp-emsA showed an incomplete larval hatching, reduced heads, and severe neuromotor defects. Furthermore, in situ hybridization revealed a spatial and temporal expression pattern that highly correlates with Rp-ems observed function. Here, Rp-ems function in R. prolixus development was validated, showing that empty-spiracles does not act as a true head-gap gene, but it is necessary for proper head development and crucial for early embryo determination and neurodevelopment. [Display omitted] • We functionally describe the empty-spiracles gene in Rhodnius prolixus • Rp-ems is maternally expressed in ovary tropharium, follicular cells, and oocyte nucleus. • Zygotic expression is located in the head region and in a body segmented pattern. • Maternal RNAi phenotype shows eggs without any embryological structure. • Zygotic Rp-ems knockdown results in embryos with cephalic and neuromotor defects. [ABSTRACT FROM AUTHOR]

Heat stress has broad effects on an organism and is an inevitable part of life. Embryos face a particular challenge when faced with heat stress - the intricate molecular processes that pattern the embryo can all be affected by heat, and the embryo lacks some of the strategies that adults can use to manage or avoid heat stress. We use Drosophila melanogaster as a model, as insects are capable of developing normally under a wide range of temperatures and are exposed to daily temperature swings as they develop. Research has focused on the heat shock pathway and the transcription of heat shock proteins as the main response to heat and heat damage. This review explores embryonic heat responses beyond the heat shock pathway. We examine the effects of heat from a biochemical standpoint, as well as highlighting other mechanisms of heat stress regulation, such as miRNA activity or other signaling pathways. We discuss how different elements of the heat stress response must be coordinated across the embryo to enable development under a wide range of temperatures. Studying heat stress in Drosophila melanogaster can be a powerful lens into how developmental systems ensure robustness to environmental factors., (Copyright © 2024. Published by Elsevier Inc.)

The serotonergic lineage (NB7-3) in the Drosophila ventral nerve cord produces six cells during neurogenesis. Four of the cells differentiate into neurons: EW1, EW2, EW3 and GW. The other two cells undergo apoptosis. This simple lineage provides an opportunity to examine genes that are required to induce or repress apoptosis during cell specification. Previous studies have shown that Notch signaling induces apoptosis within the NB7-3 lineage. The three EW neurons are protected from Notch-induced apoptosis by asymmetric distribution of Numb protein, an inhibitor of Notch signaling. In a numb 1 mutant EW2 and EW3 undergo apoptosis. The EW1 and GW neurons survive even in a numb 1 mutant background suggesting that these cells are protected from Notch-induced apoptosis by some factor other than Numb. The EW1 and GW neurons are mitotic sister cells, and uniquely express the transcription factor Hunchback. We present evidence that Hunchback prevents apoptosis in the NB7-3 lineage during normal CNS development and can rescue the two apoptotic cells in the lineage when it is ectopically expressed. We show that hunchback overexpression produces ectopic cells that express markers similar to the EW2 neuron and changes the expression pattern of the EW3 neuron to a EW2 neuron. In addition we show that hunchback overexpression can override apoptosis that is genetically induced by the pro-apoptotic genes grim and hid. [Display omitted] • Hunchback is necessary to prevent apoptosis of the GW neuron during specification of the Drosophila NB 7-3 lineage. • Ectopic Hunchback expression rescues apoptotic cells in the Drosophila NB 7-3 lineage. • Ectopic Hunchback produces ectopic serotonin neurons that resemble the EW2 neuron in the Drosophila NB 7-3 lineage. • Ectopic Hunchback can prevent apoptosis induced by the misexpression of pro-apoptotic genes in the Drosophila NB 7-3 lineage. [ABSTRACT FROM AUTHOR]

Entero-endocrine cells involved in the regulation of digestive function form a large and diverse cell population within the intestinal epithelium of all animals. Together with absorptive enterocytes and secretory gland cells, entero-endocrine cells are generated by the embryonic endoderm and, in the mature animal, from a pool of endoderm derived, self-renewing stem cells. Entero-endocrine cells share many structural/functional and developmental properties with sensory neurons, which hints at the possibility of an ancient evolutionary relationship between these two cell types. We will survey in this article recent findings that emphasize the similarities between entero-endocrine cells and sensory neurons in vertebrates and insects, for which a substantial volume of data pertaining to the entero-endocrine system has been compiled. We will then report new findings that shed light on the specification and morphogenesis of entero-endocrine cells in Drosophila. In this system, presumptive intestinal stem cells (pISCs), generated during early metamorphosis, undergo several rounds of mitosis that produce the endocrine cells and stem cells (ISCs) with which the fly is born. Clonal analysis demonstrated that individual pISCs can give rise to endocrine cells expressing different types of peptides. Immature endocrine cells start out as unpolarized cells located basally of the gut epithelium; they each extend an apical process into the epithelium which establishes a junctional complex and apical membrane specializations contacting the lumen of the gut. Finally, we show that the Drosophila homolog of ngn3, a bHLH gene that defines the entero-endocrine lineage in mammals, is expressed and required for the differentiation of this cell type in the fly gut.

Visual information processing in animals with large image forming eyes is carried out in highly structured retinotopically ordered neuropils. Visual neuropils in Drosophila form the optic lobe, which consists of four serially arranged major subdivisions; the lamina, medulla, lobula and lobula plate; the latter three of these are further subdivided into multiple layers. The visual neuropils are formed by more than 100 different cell types, distributed and interconnected in an invariant highly regular pattern. This pattern relies on a protracted sequence of developmental steps, whereby different cell types are born at specific time points and nerve connections are formed in a tightly controlled sequence that has to be coordinated among the different visual neuropils. The developing fly visual system has become a highly regarded and widely studied paradigm to investigate the genetic mechanisms that control the formation of neural circuits. However, these studies are often made difficult by the complex and shifting patterns in which different types of neurons and their connections are distributed throughout development. In the present paper we have reconstructed the three-dimensional architecture of the Drosophila optic lobe from the early larva to the adult. Based on specific markers, we were able to distinguish the populations of progenitors of the four optic neuropils and map the neurons and their connections. Our paper presents sets of annotated confocal z-projections and animated 3D digital models of these structures for representative stages. The data reveal the temporally coordinated growth of the optic neuropils, and clarify how the position and orientation of the neuropils and interconnecting tracts (inner and outer optic chiasm) changes over time. Finally, we have analyzed the emergence of the discrete layers of the medulla and lobula complex using the same markers (DN-cadherin, Brp) employed to systematically explore the structure and development of the central brain neuropil. Our work will facilitate experimental studies of the molecular mechanisms regulating neuronal fate and connectivity in the fly visual system, which bears many fundamental similarities with the retina of vertebrates.

The visceral musculature of the Drosophila intestine plays important roles in digestion as well as development. Detailed studies investigating the embryonic development of the visceral muscle exist; comparatively little is known about postembryonic development and metamorphosis of this tissue. In this study we have combined the use of specific markers with electron microscopy to follow the formation of the adult visceral musculature and its involvement in gut development during metamorphosis. Unlike the adult somatic musculature, which is derived from a pool of undifferentiated myoblasts, the visceral musculature of the adult is a direct descendant of the larval fibers, as shown by activating a lineage tracing construct in the larval muscle and obtaining labeled visceral fibers in the adult. However, visceral muscles undergo a phase of remodeling that coincides with the metamorphosis of the intestinal epithelium. During the first day following puparium formation, both circular and longitudinal syncytial fibers dedifferentiate, losing their myofibrils and extracellular matrix, and dissociating into mononuclear cells ("secondary myoblasts"). Towards the end of the second day, this process is reversed, and between 48 and 72h after puparium formation, a structurally fully differentiated adult muscle layer has formed. We could not obtain evidence that cells apart from the dedifferentiated larval visceral muscle contributed to the adult muscle, nor does it appear that the number of adult fibers (or nuclei per fiber) is increased over that of the larva by proliferation. In contrast to the musculature, the intestinal epithelium is completely renewed during metamorphosis. The adult midgut epithelium rapidly expands over the larval layer during the first few hours after puparium formation; in case of the hindgut, replacement takes longer, and proceeds by the gradual caudad extension of a proliferating growth zone, the hindgut proliferation zone (HPZ). The subsequent elongation of the hindgut and midgut, as well as the establishment of a population of intestinal stem cells active in the adult midgut and hindgut, requires the presence of the visceral muscle layer, based on the finding that ablation of this layer causes a severe disruption of both processes.

Experimental embryologists working at the turn of the 19th century suggested fundamental mechanisms of development, such as localized cytoplasmic determinants and tissue induction. However, the molecular basis underlying these processes proved intractable for a long time, despite concerted efforts in many developmental systems to isolate factors with a biological role. That road block was overcome by combining developmental biology with genetics. This powerful approach used unbiased genome-wide screens to isolate mutants with developmental defects and to thereby identify genes encoding key determinants and regulatory pathways that govern development. Two small invertebrates were the pioneers: the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Their modes of development differ in many ways, but the two together led the way to unraveling the molecular mechanisms of many fundamental developmental processes. The discovery of the grand homologies between key players in development throughout the animal kingdom underscored the usefulness of studying these small invertebrate models for animal development and even human disease. We describe developmental genetics in Drosophila and C. elegans up to the rise of genomics at the beginning of the 21st Century. Finally, we discuss themes that emerge from the histories of such distinct organisms and prospects of this approach for the future. [Display omitted] • Genome-wide mutant screens in the fly Drosophila and the worm C.elegans made major contributions to Developmental Biology. • The screens identified genes encoding key determinants and regulatory pathways that govern development. • C. elegans and Drosophila are very different invertebrate organisms with complementary strengths as models for Development. • Many regulators found in worms or flies are conserved in vertebrates and are associated with human health and disease.. [ABSTRACT FROM AUTHOR]

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement. [Display omitted] [ABSTRACT FROM AUTHOR]

Maintenance of appropriate muscle mass is crucial for physical activity and metabolism. Aging and various pathological conditions can cause sarcopenia, a condition characterized by muscle mass decline. Although sarcopenia has been actively studied, the mechanisms underlying muscle atrophy are not well understood. Thus, we aimed to investigate the role of Phosphatidylserine synthase (Pss) in muscle development and homeostasis in Drosophila. The results showed that muscle-specific Pss knockdown decreased exercise capacity and produced sarcopenic phenotypes. In addition, it increased the apoptosis rate because of the elevated reactive oxygen species production resulting from mitochondrial dysfunction. Moreover, the autophagy rate increased due to increased FoxO activity caused by reduced Akt activity. Collectively, these findings demonstrate that enhanced apoptosis and autophagy rates resulting from muscle-specific Pss knockdown jointly contribute to sarcopenia development, highlighting the key role of the PSS pathway in muscle health., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vitamin A deficiency can cause human pathologies that range from blindness to embryonic malformations. This diversity is due to the lack of two major vitamin A metabolites with very different functions: the chromophore 11- cis -retinal (vitamin A aldehyde) is a critical component of the visual pigment that mediates phototransduction, while the signaling molecule all- trans -retinoic acid regulates the development of various tissues and is required for the function of the immune system. Since animals cannot synthesize vitamin A de novo , they must obtain it either as preformed vitamin A from animal products or as carotenoid precursors from plant sources. Due to its essential role in the visual system, acute vitamin A deprivation impairs photoreceptor function and causes night blindness (poor vision under dim light conditions), while chronic deprivation results in retinal dystrophies and photoreceptor cell death. Chronic vitamin A deficiency is the leading cause of preventable childhood blindness according to the World Health Organization. Due to the requirement of vitamin A for retinoic acid signaling in development and in the immune system, vitamin A deficiency also causes increased mortality in children and pregnant women in developing countries. Drosophila melanogaster is an excellent model to study the effects of vitamin A deprivation on the eye because vitamin A is not essential for Drosophila development and chronic deficiency does not cause lethality. Moreover, genetic screens in Drosophila have identified evolutionarily conserved factors that mediate the production of vitamin A and its cellular uptake. Here, we review our current knowledge about the role of vitamin A in the visual system of mammals and Drosophila melanogaster. We compare the molecular mechanisms that mediate the uptake of dietary vitamin A precursors and the metabolism of vitamin A, as well as the consequences of vitamin A deficiency for the structure and function of the eye. [Display omitted] • Comparison of mammalian and fly models to study the role of vitamin A in vision. • Vitamin A deprivation causes similar molecular and functional defects in the eye. • Shared mechanisms for chromophore and visual pigment formation. • Mechanisms for vitamin A distribution and storage are different. • Light response of the visual pigment and role of the visual cycle also differ. [ABSTRACT FROM AUTHOR]

Iron (Fe) is used in various cellular functions, and a constant balance between its uptake, transport, storage, and use is necessary to maintain its homeostasis in the body. Changes in Fe metabolism with a consequent overload of this metal are related to neurological changes and cover a broad spectrum of diseases, mainly when these changes occur during the embryonic period. This work aimed to evaluate the effect of exposure to Fe overload during the embryonic period of Drosophila melanogaster. Progenitor flies (male and female) were exposed to ferrous sulfate (FeSO 4) for ten days in concentrations of 0.5, 1, and 5 ​mM. After mating and oviposition, the progenitors were removed and the treatment bottles preserved, and the number of daily hatches and cumulative hatching of the first filial generation (F1) were counted. Subsequently, F1 flies (separated by sex) were subjected to behavioral tests such as negative geotaxis test, open field test, grooming, and aggression test. They have evaluated the levels of dopamine (DA), serotonin (5-HT), octopamine (OA), tryptophan and tyrosine hydroxylase (TH), acetylcholinesterase, reactive species, and the levels of Fe in the progenitor flies and F1. The Fe levels of F1 flies are directly proportional to what is incorporated during the period of embryonic development; we also observed a delay in hatching and a reduction in the number of the hatch of F1 flies exposed during the embryonic period to the 5mM Fe diet, a fact that may be related to the reduction of the cell viability of the ovarian tissue of progenitor flies. The flies exposed to Fe (1 and 5 ​mM) showed an increase in locomotor activity (hyperactivity) and a significantly higher number of repetitive movements. In addition to a high number of aggressive encounters when compared to control flies. We can also observe an increase in the levels of biogenic amines DA and 5-HT and an increase in TH activity in flies exposed to Fe (1 and 5 ​mM) compared to the control group. We conclude that the hyperactive-like behavior demonstrated in both sexes by F1 flies exposed to Fe may be associated with a dysregulation in the levels of DA and 5-HT since Fe is a cofactor of TH, which had its activity increased in this study. Therefore, more attention is needed during the embryonic development period for exposure to Fe overload. [Display omitted] • Progenitors flies exposed to Fe reduced ovarian cell viability. • F1 flies exposed to Fe in the embryonic period have increased DA, 5-HT and TH. • F1 Flies exposed to Fe in the embryonic period became hyperactive. • F1 Flies exposed to Fe in the embryonic period showed repetitive movements. [ABSTRACT FROM AUTHOR]

Apoptosis is a fundamental remodeling process for most tissues during development. In this manuscript we examine a pro-apoptotic function for the Drosophila DNA binding protein Zfh-2 during development of the central nervous system (CNS) and appendages. In the CNS we find that a loss-of-function zfh-2 allele gives an overall reduction of apoptotic cells in the CNS, and an altered pattern of expression for the axonal markers 22C10 and FasII. This same loss-of-function zfh-2 allele causes specific cells in the NB7-3 lineage of the CNS that would normally undergo apoptosis to be inappropriately maintained, whereas a gain-of-function zfh-2 allele has the opposite effect, resulting in a loss of normal NB 7-3 progeny. We also demonstrate that Zfh-2 and Hunchback reciprocally repress each other's gene expression which limits apoptosis to later born progeny of the NB7-3 lineage. Apoptosis is also required for proper segmentation of the fly appendages. We find that Zfh-2 co-localizes with apoptotic cells in the folds of the imaginal discs and presumptive cuticular joints. A reduction of Zfh-2 levels with RNAi inhibits expression of the pro-apoptotic gene reaper, and produces abnormal joints in the leg, antenna and haltere. Apoptosis has previously been shown to be activated by Notch signaling in both the NB7-3 CNS lineage and the appendage joints. Our results indicate that Zfh-2 facilitates Notch-induced apoptosis in these structures. [Display omitted] • Zfh-2 facilitates Notch-mediated apoptosis during specification of the Drosophila NB 7-3 lineage. • Zfh-2 and Hunchback transcriptionally repress each other in the Drosophila NB 7-3 lineage. • Zfh-2 facilitates Notch-mediated apoptosis during development of cuticular joints in Drosophila appendages. • Zfh-2 induces reaper expression in the leg disc. • Apical constriction results in a distinct spatial patterning of Zfh-2, Notch and apoptosis fragments during disc folding. [ABSTRACT FROM AUTHOR]

Sustaining life requires efficient uptake of nutrients and conversion to useable forms. Almost everything about this process is dynamic. Nutrient availability fluctuates and changing environmental conditions impose new demands that can tip the metabolic equilibrium from biosynthesis and macromolecule storage to energy expenditure. At the same time, the organism itself changes, particularly during the rapid growth and differentiation in early development and also later in life as the adult ages. Here we review what has been learned from Drosophila melanogaster as an experimental model about the connections between external signals, signaling pathways, tissues and organs that allow animals to balance energy storage with expenditure in the face of change, both intrinsic and extrinsic. • Drosophila melanogaster is a powerful experimental system to study energy balance. • Communication between different organs/tissues is key for maintaining energy balance. • Nutritional requirements vary between different stages of organismal development. • Conserved signaling pathways link energy expenditure and storage to availability. [ABSTRACT FROM AUTHOR]

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline.

Actin filament crosslinking, bundling and molecular motor proteins are necessary for the assembly of epithelial projections such as microvilli, stereocilia, hairs, and bristles. Mutations in such proteins cause defects in the shape, structure, and function of these actin - based protrusions. One protein necessary for stereocilia formation, Myosin VIIA, is an actin - based motor protein conserved throughout phylogeny. In Drosophila melanogaster, severe mutations in the MyoVIIA homolog crinkled (ck) are "semi - lethal" with only a very small percentage of flies surviving to adulthood. Such survivors show morphological defects related to actin bundling in hairs and bristles. To better understand ck /MyoVIIA's function in bundled - actin structures, we used dominant female sterile approaches to analyze the loss of maternal and zygotic (M/Z) ck/ MyoVIIA in the morphogenesis of denticles, small actin - based projections on the ventral epidermis of Drosophila embryos. M/Z ck mutants displayed severe defects in denticle morphology – actin filaments initiated in the correct location, but failed to elongate and bundle to form normal projections. Using deletion mutant constructs, we demonstrated that both of the C - terminal MyTH4 and FERM domains are necessary for proper denticle formation. Furthermore, we show that ck /MyoVIIA interacts genetically with dusky - like (dyl) , a member of the ZPD family of proteins that links the extracellular matrix to the plasma membrane, and when mutated also disrupts normal denticle formation. Loss of either protein alone does not alter the localization of the other; however, loss of the two proteins together dramatically enhances the defects in denticle shape observed when either protein alone was absent. Our data indicate that ck /MyoVIIA plays a key role in the formation and/or organization of actin filament bundles, which drive proper shape of cellular projections. • Loss of maternal and zygotic ck /MyoVIIA disrupts denticle morphology. • ck /MyoVIIA mutant denticles fail to elongate and have multiple apices. • MyTH4 - FERM domains are necessary for actin-based protrusion formation. • Loss of ck /MyoVIIA and d yl results in severe abnormalities in denticle morphology. [ABSTRACT FROM AUTHOR]

Transdifferentiation is a conversion of an already differentiated cell type into another cell type without the involvement of stem cells. This transition is well described in the case of vertebrate immune cells, as well as in Drosophila melanogaster , which therefore serves as a suitable model to study the process in detail. In the Drosophila larva, the latest single-cell sequencing methods enabled the clusterization of the phagocytic blood cells, the plasmatocytes, which are capable of transdifferentiation into encapsulating cells, the lamellocytes. Here we summarize the available data of the past years on the plasmatocyte-lamellocyte transition, and make an attempt to harmonize them with transcriptome-based blood cell clustering to better understand the underlying mechanisms of transdifferentiation in Drosophila , and in general. Image 1 • We summarize mechanisms which govern hemocyte differentiation in Drosophila. • We review new methods which allowed the re-evaluation of hemocyte classification. • The new advances in hemocyte transdifferentiation are discussed. [ABSTRACT FROM AUTHOR]

The regulation of formation of the Drosophila heart by the Nkx 2.5 homologue Tinman is a key event during embryonic development. In this study, we identify the highly conserved transcription cofactor Akirin as a key factor in the earliest induction of tinman by the Twist transcription cofactor. akirin mutant embryos display a variety of morphological defects in the heart, including abnormal spacing between rows of aortic cells and abnormal patterning of the aortic outflow tract. akirin mutant embryos have a greatly reduced level of tinman transcripts, together with a reduction of Tinman protein in the earliest stages of cardiac patterning. Further, akirin mutants have reduced numbers of Tinman-positive cardiomyoblasts, concomitant with disrupted patterning and organization of the heart. Finally, despite the apparent formation of the heart in akirin mutants, these mutant hearts exhibit fewer coordinated contractions in akirin mutants compared with wild-type hearts. These results indicate that Akirin is crucial for the first induction of tinman by the Twist transcription factor, and that the success of the cardiac patterning program is highly dependent upon establishing the proper level of tinman at the earliest steps of the cardiac developmental pathway. • Akirin mutants have highly reduced levels of tinman expression at the earliest steps of cardiac development. • Akirin mutant hearts display morphological abnormalities and reduced numbers of Tinman-positive cardiomyoblasts. • Akirin mutant hearts exhibit significantly reduced numbers of contractions compared to wild-type hearts. [ABSTRACT FROM AUTHOR]

Wnt genes encode secreted ligands that play many important roles in the development of metazoans. There are thirteen known Wnt gene subfamilies and seven of these are represented in Drosophila melanogaster. While wingless (wg) is the best understood and most widely studied Wnt gene in Drosophila , the functions of many of the other Drosophila Wnt genes are less well understood. For example, relatively little is known about Wnt6 , which is an ancient paralog of wg and they form a conserved Wnt cluster together with Wnt9 (Dwnt4) and Wnt10. Wg and Wnt6 encode similar proteins and exhibit overlapping expression in several tissues during development. Both wg and Wnt6 were previously shown to regulate the development of maxillary palps, important olfactory organs in flies, but it remained unclear how these two ligands may combine to carry out specific functions and how this is regulated. Here, we have further analysed Wnt6 function in the context of maxillary palp development. Surprisingly, we found that Wnt6 does not appear to be necessary for development of maxillary palps. While a deletion of the 5' region of Wnt6 results in very small maxillary palps, we show that this effect is more likely to be a consequence of removing cis - regulatory elements that may regulate wg expression in this tissue rather than through the loss of Wnt6 function. Although, we cannot completely exclude the possibility that Wnt6 may subtly regulate maxillary palp development in combination with wg , our analysis of Wnt6 loss of function mutants suggests this ligand plays a more general role in regulating growth during development. Taken together our results provide new insights into maxillary palp formation and Wnt6 functions in Drosophila , and further evidence for a complex cis-regulatory landscape in the Wnt9-wg-Wnt6-Wnt10 cluster, which may help explain its evolutionary conservation. • Wnt6 appears to regulate general growth during Drosophila development and time to pupariation is delayed in Wnt6 mutants. • Wg and Wnt6 are expressed in the maxillary palps but only the former is required for the development of these organs. • The Wnt6 locus may host cis-regulatory elements required for wg expression in the maxillary palps. • Distal and shared enhancers may contribute to the conservation and functionality of the Wnt9-wg-Wnt6-Wnt10 cluster. [ABSTRACT FROM AUTHOR]

scarecrow (scro) gene encodes a Drosophila homolog of mammalian Nkx2. 1 that belongs to an evolutionally conserved NK2 family. Nkx2. 1 has been well known for its role in the development of hypothalamus, lung, thyroid gland, and brain. However, little is known about biological roles of scro. To understand scro functions, we generated two types of knock-in mutant alleles, substituting part of either exon-2 or exon-3 for EGFP (or Gal4) by employing the CRISPR/Cas9 genome editing tool. Using these mutations, we characterized spatio-temporal expression patterns of the scro gene and its mutant phenotypes. Homozygous knock-in mutants are lethal during embryonic and early larval development. In developing embryos, scro is exclusively expressed in the pharyngeal primordia and numerous neural clusters in the central nervous system (CNS). In postembryonic stages, the most prominent scro expression is detected in the larval and adult optic lobes, suggesting that scro plays a role for the development and/or function of this tissue type. Notch signaling is the earliest factor known to act for the development of the optic lobe. scro mutants lacked mitotic cells and Delta expression in the optic anlagen, and showed altered expression of several proneural and neurogenic genes including Delta and Notch. Furthermore, scro mutants showed grossly deformed neuroepithelial (NE) cells in the developing optic lobe and severely malformed adult optic lobes, the phenotypes of which are shown in Notch or Delta mutants, suggesting scro acting epistatic to the Notch signaling. From these data together, we propose that scro plays an essential role for the development of the optic lobe, possibly acting as a regional specification factor. • Nk2.1 / scro mutants were generated by CRISPR/Cas9-mediated knock-in technique. • Loss of scro function leads to lethality at the second larval stage and severe disruption of brain structure. • scro is highly expressed in the medulla and lamina of optic lobe in postembryonic stages. • scro interacts with proneural and neurogenic genes during brain development. [ABSTRACT FROM AUTHOR]

Rab11, a member of Rab-GTPase family, and a marker of recycling endosomes has been reported to be involved in the differentiation of various tissues in Drosophila. Here we report a novel role of Rab11 in the differentiation of stellate cells via the non-canonical Notch pathway in Malpighian tubules. During Malpighian tubule development caudal visceral mesodermal cells intercalate into the epithelial tubule of ectodermal origin consisting of principal cells, undergo mesenchymal to epithelial transition and differentiate into star shaped stellate cells in adult Malpighian tubule. Two transcription factors, Teashirt and Cut (antagonistic to each other) are known to be expressed in stellate cells and principal cells, respectively, from early stages of development and serve as markers for these cells. Inhibition of Rab11 function or over-expression of activated Notch in stellate cells resulted in the expression of Cut that leads to down-regulation of Teashirt or vice-versa that leads to hampered differentiation of stellate cells. The stellate cells do not transform to star/bar shaped and remain in mesenchymal state in adult Malpighian tubule. Over-expression of Deltex, which plays important role in non-canonical Notch signaling pathway, shows similar phenotype of stellate cells as seen in individuals with down-regulated Rab11, while down-regulation of Deltex in genetic background of Rab11 RNAi rescues Teashirt expression and shape of stellate cells. Our experiments suggest that an inhibition or reduction of Rab11 function in stellate cells results in the faulty recycling of Notch receptors to plasma membrane as they accumulate in early and late endosomes, leading to Deltex mediated non-canonical Notch activation. • Rab11 is indispensable for maintenance of shape and required for MET of SCs. • Rab11 genetically interact with Deltex during SCs morphogenesis. • Rab11 is responsible for proper localization of Notch on early and late endosomes in SCs. [ABSTRACT FROM AUTHOR]

Protection of the germline from somatic differentiation programs is crucial for germ cell development. In many animals, whose germline development relies on the maternally inherited germ plasm, such protection in particular at early stages of embryogenesis is achieved by maternally localized global transcriptional repressors, such as PIE-1 of Caenorhabditis elegans , Pgc of Drosophila melanogaster and Pem of ascidians. However, zygotic gene expression starts in later germline cells eventually and mechanisms by which somatic gene expression is selectively kept under repression in the transcriptionally active cells are poorly understood. By using the ascidian species Halocynthia roretzi , we found that H3K27me3, a repressive transcription-related chromatin mark, became enriched in germline cells starting at the 64-cell stage when Pem protein level and its contribution to transcriptional repression decrease. Interestingly, inhibition of H3K27me3 together with Pem knockdown resulted in ectopic expression in germline cells of muscle developmental genes Muscle actin (MA4) and Snail , and of Clone 22 (which is expressed in all somatic but not germline cells), but not of other tissue-specific genes such as the notochord gene Brachyury , the nerve cord marker ETR-1 and a heart precursor gene Mesp , at the 110-cell stage. Importantly, these ectopically expressed genes are normally expressed in the germline sister cells (B7.5), the last somatic lineage separated from the germline. Also, the ectopic expression of MA4 was dependent on a maternally localized muscle determinant Macho-1. Taken together, we propose that H3K27me3 may be responsible for selective transcriptional repression for somatic genes in later germline cells in Halocynthia embryos and that the preferential repression of germline sister-lineage genes may be related to the mechanism of germline segregation in ascidian embryos, where the germline is segregated progressively by successive asymmetric cell divisions during cell cleavage stages. Together with findings from C. elegans and D. melanogaster , our data for this urochordate animal support the proposal for a mechanism, conserved widely throughout the animal kingdom, where germline transcriptional repression is mediated initially by maternally localized factors and subsequently by a chromatin-based mechanism. • H3K27me3 becomes enriched in ascidian germline cells from the 64-cell stage onwards. • Maternal germline silencer Pem suppresses RNAPII activity through the 110-cell stage. • H3K27me3- and Pem-deficient germline mis-expresses its sister muscle-lineage genes. • The mis-expression of muscle genes depends on a maternal determinant, Macho-1. [ABSTRACT FROM AUTHOR]

Recently, it was shown that anterior-posterior patterning genes in the red flour beetle Tribolium castaneum are expressed sequentially in waves. However, in the fruit fly Drosophila melanogaster , an insect with a derived mode of embryogenesis compared to Tribolium , anterior-posterior patterning genes quickly and simultaneously arise as mature gene expression domains that, afterwards, undergo slight posterior-to-anterior shifts. This raises the question of how a fast and simultaneous mode of patterning, like that of Drosophila , could have evolved from a rather slow sequential mode of patterning, like that of Tribolium. In this paper, we propose a mechanism for this evolutionary transition based on a switch from a uniform to a gradient-mediated initialization of the gap gene cascade by maternal Hb. The model is supported by computational analyses and experiments. • Model: Initializing a genetic cascade by a gradient induces simultaneous patterning. • The model recapitulates various reported phenotypes in Drosophila and Tribolium. • The model suggests a smooth evolutionary path for segmentation modes in insects. [ABSTRACT FROM AUTHOR]

Proper genetic and epigenetic regulation is necessary to maintain the identity and integrity of cells. Enzymes involved in post-transcriptional modifications of histones are key factors in epigenetic mechanisms. Such modifications are also gaining importance for their role in growth and development of cancer. SETDB1 catalyzes the epigenetic mark of lysine-9 methylation of histone-3. In this study, we explored the role of SETDB1 in Drosophila hematopoiesis. We show that SETDB1 controls the differentiation of matured blood cells in wandering third instar larvae. There are three matured blood cells in wild type Drosophila melanogaster : plasmatocytes, crystal cells and lamellocytes. We found that loss-of-function mutants of SETDB1 show hematopoietic defects; increased blood cell proliferation, decreased number of crystal cells, greater differentiation of blood cells into lamellocytes, dysplasia of the anterior lobes of lymph gland and presence of hematopoietic tumors. Cell type specific knockdown of SETDB1 provided similar phenotype i.e., decreased number of crystal cells and an increase in lamellocyte differentiation. In animals with loss of function of SETDB1, Notch pathway was downregulated. Further, over-expression of SETDB1 in blood cells resulted in an increase in the number of crystal cells. This increase is accompanied with an increase in the number of NotchICD expressing cells. We therefore performed genetic rescue using UAS-GAL4 system to rescue loss of function SETDB1 mutants. Our data show that the rescued larvae carrying a wild type copy of SETDB1 in mutant background are devoid of blood tumors. We have identified a novel dual function of SETDB1 methylatransferase as a critical regulator of two of the matured hemocytes, crystal cells and lamellocytes. We propose a novel role of SETDB1 in modulating the differentiation of crystal cells and lamellocytes from a common progenitor and underscore the importance of SETDB1 in Drosophila blood tumor suppression. • SETDB1 regulates crystal cell differentiation: Over-expression of SETDB1 increases the crystal cell number (≤5% in the wild type) i.e. when SETDB1 levels are increased in the blood cells we detected an increase in the number of crystal cells. Our results demonstrate that SETDB1 has a hold on maintaining the number of crystal number in wild type flies. • SETDB1 controls lamellocyte differentiation: Loss-of-function of SETDB1 leads to increased differentiation of blood cells into lamellocytes (<1% in the wild type) i.e. regulates progenitor differentiation into lamellocytes. • SETDB1 is a blood tumor suppressor: We observed blood tumors both, in the circulating hemolymph and in the developing hematopoietic organ, the lymph gland. These tumors are exclusively composed of blood cells i.e. abnormal plasmatocytes and also lamellocytes. Genetic rescue with the wild type SETDB1 expression in the loss-of-function mutants of SETDB1 relieves it from both lamellocyte and blood tumor formation. Therefore, our results clearly establish the novel function of SETDB1 as a blood tumor suppressor. [ABSTRACT FROM AUTHOR]

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster model of eIF6 upregulation, leading to a boost in general translation and the shut-down of the ecdysone biosynthetic pathway. Indeed, translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo , eIF6-driven alterations delayed Programmed Cell Death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation. • eIF6 high levels result in heightened general translation. • Ecdysone biosynthesis and general translational rate are inversely correlated. • Apoptosis is delayed by ecdysone biosynthesis shut-down during development. [ABSTRACT FROM AUTHOR]

The terminal cells of the tracheal epithelium in Drosophila melanogaster are one of the few known cell types that undergo subcellular morphogenesis to achieve a stable, branched shape. During the animal's larval stages, the cells repeatedly sprout new cytoplasmic processes. These grow very long, wrapping around target tissues to which the terminal cells adhere, and are hollowed by a gas-filled subcellular tube for oxygen delivery. Our understanding of this ramification process remains rudimentary. This review aims to provide a comprehensive summary of studies on terminal cells to date, and attempts to extrapolate how terminal branches might be formed based on the known genetic and molecular components. Next to this cell-intrinsic branching mechanism, we examine the extrinsic regulation of terminal branching by the target tissue and the animal's environment. Finally, we assess the degree of similarity between the patterns established by the branching programs of terminal cells and other branched cells and tissues from a mathematical and conceptual point of view. • Branch growth in terminal cells depends on concomittant growth of a subcellular tube. • Existing evidence allows synthesising a working model of terminal branch formation. • Terminal cells sense hypoxia like most cells but respond uniquely. • The classic model of terminal branch guidance is inconsistent with newer evidence. [ABSTRACT FROM AUTHOR]

The regenerative process after tissue damage relies on a variety of cellular responses that includes compensatory cell proliferation and cell fate re-specification. The identification of the signalling networks regulating these cellular events is a central question in regenerative biology. Tissue regeneration models in Drosophila have shown that two of the signals that play a fundamental role during the early stages of regeneration are the c-Jun N-terminal kinase (JNK) and JAK/STAT signalling pathways. These pathways have been shown to be required for controlling regenerative proliferation, however their contribution to the processes of cellular reprogramming and cell fate re-specification that take place during regeneration are largely unknown. Here, we present evidence for a previously unrecognised function of the cooperative activities of JNK and JAK/STAT signalling pathways in inducing loss of cell fate specification in imaginal discs. We show that co-activation of these signalling pathways induces both the cell fate changes in injured areas, as well as in adjacent cells. We have also found that this function relies on the activity of the Caspase initiator encoded by the gene dronc . [ABSTRACT FROM AUTHOR]