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The epidermal cells of insects are polarized epithelial cells that play a pivotal role in the insect's molting process. Sinuous, a pivotal structural protein involved in the formation of septate junctions among epithelial cells, is essential for its physiological function. In this study, to determine whether sinuous participates in the regulation of insect molting, we identified the sinuous gene, Lmsinu, in Locusta migratoria, which encodes a protein belonging to the claudin family and shares 62.6% identity with Drosophila's sinuous protein. Lmsinu is expressed in multiple tissues, and its expression level in the integument significantly increases prior to molting. Knockdown of Lmsinu in L. migratoria results in larval mortality during molting. Furthermore, hematoxylin and eosin and chitin staining demonstrate that the downregulation of Lmsinu led to a prolonged degradation process of the old cuticle during the molting process. Electron microscopy analysis further revealed that knockdown of Lmsinu disrupts the formation of septate junctions among epidermal cells, which are a monolayer of polarized epithelial cells, which may hinder the functionality of epidermal cells during the process of molting. In summary, these findings suggest that Lmsinu plays a role in nymph molting by regulating the formation of septate junctions among epidermal cells. [ABSTRACT FROM AUTHOR]

In the adult Drosophila midgut, basal intestinal stem cells give rise to enteroblasts that integrate into the epithelium as they differentiate into enterocytes. Integrating enteroblasts must generate a new apical domain and break through the septate junctions between neighbouring enterocytes, while maintaining barrier function. We observe that enteroblasts form an apical membrane initiation site (AMIS) when they reach the septate junction between the enterocytes. Cadherin clears from the apical surface and an apical space appears between above the enteroblast. New septate junctions then form laterally with the enterocytes and the AMIS develops into an apical domain below the enterocyte septate junction. The enteroblast therefore forms a pre-assembled apical compartment before it has a free apical surface in contact with the gut lumen. Finally, the enterocyte septate junction disassembles and the enteroblast/pre-enterocyte reaches the gut lumen with a fully formed brush border. The process of enteroblast integration resembles lumen formation in mammalian epithelial cysts, highlighting the similarities between the fly midgut and mammalian epithelia.

Septate junctions (SJs) serve as occluding barriers in invertebrate epithelia. In Drosophila, at least 30 genes are required for the formation or maintenance of SJs. Interestingly, loss-of-function mutations in core SJ components are embryonic lethal, with defects in developmental events such as head involution and dorsal closure (DC) that occur prior to the formation of a mature SJ, indicating a role for these proteins in mid-embryogenesis independent of their occluding function. To understand this novel function in development, we examined loss-of-function mutations in three core SJ proteins during the process of DC. DC occurs during mid-embryogenesis to seal a dorsal gap in the epidermis following germ band retraction. Closure is driven by contraction of the extraembryonic amnioserosa cells that temporarily cover the dorsal surface and by cell shape changes (elongation) of lateral epidermal cells that bring the contralateral sheets together at the dorsal midline. Using live imaging and examination of fixed tissues, we show that early events in DC occur normally in SJ mutant embryos, but during later closure, coracle, Macroglobulin complement-related and Neurexin-IV mutant embryos exhibit slower rates of closure and display aberrant cells shapes in the dorsolateral epidermis, including dorsoventral length and apical surface area. SJ mutant embryos also show mild defects in actomyosin structures along the leading edge, but laser cutting experiments suggest similar tension and viscoelastic properties in SJ mutant versus wild type epidermis. In a high percentage of SJ mutant embryos, the epidermis tears free from the amnioserosa near the end of DC and live imaging and immunostaining reveal reduced levels of E-cadherin, suggesting that defective adhesion may be responsible for these tears. Supporting this notion, reducing E-cadherin by half significantly enhances the penetrance of DC defects in coracle mutant embryos.

The blood–brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA’s molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. Based on these findings, we propose that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical to maintain tissue size and brain homeostasis during organogenesis.

Regeneration of Drosophila imaginal discs, larval precursors to adult tissues, activates a regeneration checkpoint that coordinates regenerative growth with developmental progression. This regeneration checkpoint results from the release of the relaxin-family peptide Dilp8 from regenerating imaginal tissues. Secreted Dilp8 protein is detected within the imaginal disc lumen, in which it is separated from its receptor target Lgr3, which is expressed in the brain and prothoracic gland, by the disc epithelial barrier. Here, we demonstrate that following damage the imaginal disc epithelial barrier limits Dilp8 signaling and the duration of regeneration checkpoint delay.We also find that the barrier becomes increasingly impermeable to the transepithelial diffusion of labeled dextran during the second half of the third instar. This change in barrier permeability is driven by the steroid hormone ecdysone and correlates with changes in localization of Coracle, a component of the septate junctions that is required for the late-larval impermeable epithelial barrier. Based on these observations, we propose that the imaginal disc epithelial barrier regulates the duration of the regenerative checkpoint, providing a mechanism by which tissue function can signal the completion of regeneration. [ABSTRACT FROM AUTHOR]

Epithelial tissues are fundamental for the establishment and maintenance of different body compartments in multicellular animals. To achieve this specific task epithelial sheets secrete an apical extracellular matrix for tissue strength and protection and they organize a transepithelial barrier function, which is mediated by tight junctions in vertebrates or septate junctions in invertebrates. Here, we show that the bicistronic gene würmchen is functionally expressed in epithelial tissues. CRISPR/Cas9-mediated mutations in both coding sequences reveal two essential polypeptides, Würmchen1 and Würmchen2, which are both necessary for normal epithelial tissue development. Würmchen1 represents a genuine septate junction core component. It is required during embryogenesis for septate junction organization, the establishment of a transepithelial barrier function, distinct cellular transport processes and tracheal system morphogenesis. Würmchen2 is localized in the apical membrane region of epithelial tissues and in a central core of the tracheal lumen during embryogenesis. It is essential during the later larval development. • The bicistronic gene würmchen (wrm) of Drosophila encodes two proteins, Wrm1 and Wrm2. • Wrm1 represents an essential septate junction core component. • Wrm2 is a member of the small open reading frame-encoded peptides. • Wrm2 is localized in the apical membrane region of epithelial tissues. [ABSTRACT FROM AUTHOR]

The septate junction (SJ) provides an occluding function for epithelial tissues in invertebrate organisms. This ability to seal the paracellular route between cells allows internal tissues to create unique compartments for organ function and endows the epidermis with a barrier function to restrict the passage of pathogens. Over the past twenty-five years, numerous investigators have identified more than 30 proteins that are required for the formation or maintenance of the SJs in Drosophila melanogaster, and have determined many of the steps involved in the biogenesis of the junction. Along the way, it has become clear that SJ proteins are also required for a number of developmental events that occur throughout the life of the organism. Many of these developmental events occur prior to the formation of the occluding junction, suggesting that SJ proteins possess non-occluding functions. In this review, we will describe the composition of SJs, taking note of which proteins are core components of the junction versus resident or accessory proteins, and the steps involved in the biogenesis of the junction. We will then elaborate on the functions that core SJ proteins likely play outside of their role in forming the occluding junction and describe studies that provide some cell biological perspectives that are beginning to provide mechanistic understanding of how these proteins function in developmental contexts.

• Mayfly nymphs were exposed to salt contaminated water. • Ionoregulatory homeostasis was disturbed. • Significant changes in ion transport occurred in the tracheal gills. • Expression and activities of ionomotive enzymes were unaltered in the tracheal gills. • The morphology of septate junctions in the tracheal gills is altered such that these junctions would be leakier. The impact of freshwater (FW) salinization on osmoregulation as well as tracheal gill morphology and function was examined in nymphs of the mayfly Hexagenia rigida following exposure to salt contaminated water (SCW, 7.25 g/l NaCl) for a 7-day period. Ionoregulatory homeostasis was perturbed in SCW exposed H. rigida nymphs as indicated by increased hemolymph Na+, K+ and Cl− levels as well as hemolymph pH and water content. Despite this, SCW did not alter gill Na+-K+-ATPase (NKA) or V-type H+-ATPase (VA) activity. In addition, NKA and VA immunolocalization in gill ionocytes did not show alterations in enzyme location or changes in ionocyte abundance. The latter observation was confirmed using scanning electron microscopy (SEM) to examine exposed tracheal gill ionocyte numbers. Ionocyte surface morphometrics also revealed that SCW did not change individual ionocyte surface area or ionocyte fractional surface area. Nevertheless, analysis of Na+ movement across the tracheal gill of mayfly nymphs using scanning ion-selective electrode technique indicated that FW nymphs acquired Na+ from surrounding water, while tracheal gills of SCW nymphs had the capacity to secrete Na+. Because Na+ secretion across the gill of SCW-exposed animals occurred in the absence of any change in (1) NKA and VA activity or (2) ionocyte numbers/surface exposure, it was reasoned that Na+ movement across the gill of SCW animals may be occurring, at least in part, through the paracellular pathway. The ultrastructure of tracheal gill septate junctions (SJs) supported this idea as they exhibited morphological alterations indicative of a leakier pathway. Data provide a first look at alterations in osmoregulatory mechanisms that allow H. rigida nymphs to tolerate sub-lethal salinization of their surroundings. [ABSTRACT FROM AUTHOR]

Abstract The use of oral insecticidal molecules (small molecules, peptides, dsRNA) via spray or plant mediated applications represents an efficient way to manage damaging insect species. With the exception of Bt toxins that target the midgut epithelium itself, most of these compounds have targets that lie within the hemocoel (body) of the insect. Because of this, one of the greatest factors in determining the effectiveness of an oral insecticidal compound is its ability to traverse the gut epithelium and enter the hemolymph. However, for many types of insecticidal compounds, neither the pathway taken across the gut nor the specific genes which influence uptake are fully characterized. Here, we review how different types of insecticidal compounds enter or cross the midgut epithelium through passive (diffusion) or active (transporter based, endocytosis) routes. A deeper understanding of how insecticidal molecules cross the gut will help to best utilize current insecticides and also provide for more rational design of future ones. Graphical abstract Image 1 Highlights • The ability of insecticidal molecules to cross the insect midgut is vital to their ability to kill insects. • Transporter proteins likely play a role in the penetration of small molecules across the gut but remain understudied. • A number of studies have identified the molecular components of protein uptake including receptors on the apical membrane. • Much still remains unclear about the fate of dsRNA once it is taken up into midgut cells. [ABSTRACT FROM AUTHOR]

The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood–brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster. Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction.

The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis.

Drosophila CrebA facilitates high-level secretion by transcriptional upregulation of the protein components of the core secretory machinery. In CrebA mutant embryos, both salivary gland (SG) morphology and epidermal cuticle secretion are abnormal, phenotypes similar to those observed with mutations in core secretory pathway component genes. Here, we examine the cellular defects associated with CrebA loss in the SG epithelium. Apically localized secretory vesicles are smaller and less abundant, consistent with overall reductions in secretion. Unexpectedly, global mislocalization of cellular organelles and excess membrane accumulation in the septate junctions (SJs) are also observed. Whereas mutations in core secretory pathway genes lead to organelle localization defects similar to those of CrebA mutants, they have no effect on SJ-associated membrane. Mutations in tetraspanin genes, which are normally repressed by CrebA, have mild defects in SJ morphology that are rescued by simultaneous CrebA loss. Correspondingly, removal of several tetraspanins gives partial rescue of the CrebA SJ phenotype, supporting a role for tetraspanins in SJ organization.

Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata., (© 2023 Wiley Periodicals LLC.)

In epithelial tissue, new cell-cell junctions are formed upon cytokinesis. To understand junction formation during cytokinesis, we explored de novo formation of tricellular septate junctions (TCJs) in Drosophila epithelium. We found that upon midbody formation, the membranes of the two daughter cells and of the neighbouring cells located below the adherens junction (AJ) remain entangled in a 4-cell structure apposed to the midbody. The septate junction protein Discs-Large and components of the TCJ, Gliotactin and Anakonda accumulate in this 4-cell structure. Subsequently, a basal movement of the midbody parallels the detachment of the neighbouring cell membranes from the midbody, the disengagement of the daughter cells from their neighbours and the reorganisation of TCJs between the two daughter cells and their neighbouring cells. While the movement of midbody is independent of the Alix and Shrub abscission regulators, the loss of Gliotactin or Anakonda function impedes both the resolution of the connection between the daughterneighbour cells and midbody movement. TCJ proteins therefore control an additional step of cytokinesis necessary for the disentanglement of the daughter cells from their neighbours during cytokinesis. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Unfavorable TBI outcomes result from primary mechanical injuries to the brain and ensuing secondary non-mechanical injuries that are not limited to the brain. Our genome-wide association study of Drosophila melanogaster revealed that the probability of death following TBI is associated with single nucleotide polymorphisms in genes involved in tissue barrier function and glucose homeostasis. We found that TBI causes intestinal and blood–brain barrier dysfunction and that intestinal barrier dysfunction is highly correlated with the probability of death. Furthermore, we found that ingestion of glucose after a primary injury increases the probability of death through a secondary injury mechanism that exacerbates intestinal barrier dysfunction. Our results indicate that natural variation in the probability of death following TBI is due in part to genetic differences that affect intestinal barrier dysfunction.

The pleated septate junction (pSJ), an ancient structure for cell-cell contact in invertebrate epithelia, has protein components that are found in three more-recent junctional structures, the neuronal synapse, the paranodal region of the myelinated axon and the vertebrate epithelial tight junction. These more-recent structures appear to have evolved through alterations of the ancestral septate junction. During its formation in the developing animal, the pSJ exhibits plasticity, although the final structure is extremely robust. Similar to the immature pSJ, the synapse and tight junctions both exhibit plasticity, and we consider evidence that this plasticity comes at least in part from the interaction of members of the immunoglobulin cell adhesion molecule superfamily with highly regulated membraneassociated guanylate kinases. This plasticity regulation probably arose in order to modulate the ancestral pSJ and is maintained in the derived structures; we suggest that it would be beneficial when studying plasticity of one of these structures to consider the literature on the others. Finally, looking beyond the junctions, we highlight parallels between epithelial and synaptic membranes, which both showa polarized distribution of many of the same proteins - evidence that determinants of apicobasal polarity in epithelia also participate in patterning of the synapse. [ABSTRACT FROM AUTHOR]

The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood–brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster. Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction. [ABSTRACT FROM AUTHOR]

Invertebrate diversity and architecture is immense. This is achieved by the organization and function of four tissue types found in most metazoan phyla-epithelial, connective, muscle and nervous tissue. Epithelial tissue is found in all extant animals (parazoan and metazoan alike). Epithelial cells form cellular sheets that cover internal or external surfaces and regulate the passage of material between separated compartments. The transepithelial movement of biological material between compartments can occur across the transcellular pathway (i.e. across cells) or the paracellular pathway (i.e. between cells) and the latter is regulated by occluding junctions that typically link cells in a subapical domain. In this review, information on occluding junctions of invertebrate epithelia is consolidated and discussed in the context of morphology, ultrastructure and physiology. In addition, an overview of what is currently known about invertebrate occluding junction proteins and their role in maintaining the integrity of invertebrate epithelia and regulating the barrier properties of these tissues is presented. [ABSTRACT FROM AUTHOR]

Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. Here, we report the identification of two novel SJ components, Pasiflora1 and Pasiflora2, through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are coexpressed in embryonic epithelia and glia and are required cellautonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments we demonstrate that pasiflora proteins are core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions. [ABSTRACT FROM AUTHOR]

Epithelial tissues separate body compartments with different compositions. Tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates control the paracellular flow of molecules between these compartments. This epithelial barrier function of TJs and SJs must be stably maintained in tissue morphogenesis during cell proliferation and cell movement. Here, we show that Bark beetle (Bark), a putative transmembrane scavenger receptor-like protein, is essential for the maturation but not the establishment of SJs in Drosophila . Embryos that lack bark establish functional SJs, but due to rudimentary septae formation during subsequent embryonic development, these become non-functional. Furthermore, cell adhesion is impaired at the lateral cell membrane and the core protein complexes of SJs are mis-localised, but appear to form otherwise normally in such embryos. We propose a model in which Bark acts as a scaffold protein that mediates cell adhesion and mounting of SJ core complexes during cell rearrangement in tissue morphogenesis. [ABSTRACT FROM AUTHOR]

Efficient neuronal conductance requires that axons are insulated by glial cells. For this, glial membranes need to wrap around axons. Invertebrates show a relatively simple extension of glial membranes around the axons, resembling Remak fibers formed by Schwann cells in the mammalian peripheral nervous system. To unravel the molecular pathways underlying differentiation of glial cells that provide axonal wrapping, we are using the genetically amenable Drosophila model. At the end of larval life, the wrapping glia differentiates into very large cells, spanning more than 1 mm of axonal length. The extension around axonal membranes is not influenced by the caliber of the axon or its modality. Using cell type specific gene knockdown we show that the extension of glial membranes around the axons is regulated by an autocrine activation of the EGF receptor through the neuregulin homolog Vein. This resembles the molecular mechanism employed during cell-autonomous reactivation of glial differentiation after injury in mammals. We further demonstrate that Vein, produced by the wrapping glia, also regulates the formation of septate junctions in the abutting subperineurial glia. Moreover, the wrapping glia indirectly controls the proliferation of the perineurial glia. Thus, the wrapping glia appears center stage to orchestrate the development of the different glial cell layers in a peripheral nerve. [ABSTRACT FROM AUTHOR]

Septate junctions (SJs) are specialized intercellular junctions that function as permeability barriers to restrict the free diffusion of solutes through the paracellular routes in invertebrate epithelia. SJs are subdivided into several morphological types that vary among different animal phyla. In several phyla, different types of SJ have been described in different epithelia within an individual. Arthropods have two types of SJs: pleated SJs (pSJs) and smooth SJs (sSJs), found in ectodermally and endodermally derived epithelia, respectively. Several lines of Drosophila research have identified and characterized a large number of pSJ-associated proteins. Two sSJ-specific proteins have been recently reported. Molecular dissection of SJs in Drosophila and animals in other phyla will lead to a better understanding of the functional differences among SJ types and of evolutionary aspects of these permeability barriers. [ABSTRACT FROM AUTHOR]

Fitness decreases associated with inbreeding depression often become more pronounced in a stressful environment. The functional genomic causes of these inbreeding-by-environment (I × E) interactions, and of inbreeding depression in general, are poorly known. To further our understanding of I × E interactions, we performed a genome-wide gene expression study of a single inbred line that suffers from temperature-sensitive lethality. We confirmed that increased differential expression between the thermosensitive line and the control line occurs at the restrictive temperature. This demonstrates that I × E interactions in survival are reflected in similar I × E interactions at the gene expression level. To make an impression of the cellular response associated with the lethal effect, we analysed all functional annotation terms that were overrepresented among the differentially expressed genes. Some sets of differentially expressed genes function in the general stress response, and these are more likely to also be differentially expressed in other studies of inbreeding, inbreeding depression, immunity and heat stress. Other sets of differentially expressed genes are shared with studies of gene expression in inbred lines, but not studies of the response to extrinsic stress, and represent a general transcriptomic signature of inbreeding. Finally, some sets of genes have an annotation that is not reported in other studies. These we consider to be candidates for the genes harbouring the mutations responsible for the thermosensitive phenotype, as these mutations are expected to be unique to this line. These genes may also serve as candidate QTL in studies of thermal tolerance and heat resistance. [ABSTRACT FROM AUTHOR]

In the adult Drosophila midgut, basal intestinal stem cells give rise to enteroblasts that integrate into the epithelium as they differentiate into enterocytes. Integrating enteroblasts must generate a new apical domain and break through the septate junctions between neighbouring enterocytes, while maintaining barrier function. We observe that enteroblasts form an apical membrane initiation site (AMIS) when they reach the septate junction between the enterocytes. Cadherin clears from the apical surface and an apical space appears between above the enteroblast. New septate junctions then form laterally with the enterocytes and the AMIS develops into an apical domain below the enterocyte septate junction. The enteroblast therefore forms a pre-assembled apical compartment before it has a free apical surface in contact with the gut lumen. Finally, the enterocyte septate junction disassembles and the enteroblast/pre-enterocyte reaches the gut lumen with a fully formed brush border. The process of enteroblast integration resembles lumen formation in mammalian epithelial cysts, highlighting the similarities between the fly midgut and mammalian epithelia., Competing Interests: JC, DS No competing interests declared, (© 2022, Chen and St Johnston.)

Septate junctions (SJs) serve as occluding barriers in invertebrate epithelia. In Drosophila , at least 30 genes are required for the formation or maintenance of SJs. Interestingly, loss-of-function mutations in core SJ components are embryonic lethal, with defects in developmental events such as head involution and dorsal closure (DC) that occur prior to the formation of a mature SJ, indicating a role for these proteins in mid-embryogenesis independent of their occluding function. To understand this novel function in development, we examined loss-of-function mutations in three core SJ proteins during the process of DC. DC occurs during mid-embryogenesis to seal a dorsal gap in the epidermis following germ band retraction. Closure is driven by contraction of the extraembryonic amnioserosa cells that temporarily cover the dorsal surface and by cell shape changes (elongation) of lateral epidermal cells that bring the contralateral sheets together at the dorsal midline. Using live imaging and examination of fixed tissues, we show that early events in DC occur normally in SJ mutant embryos, but during later closure, coracle , Macroglobulin complement-related and Neurexin-IV mutant embryos exhibit slower rates of closure and display aberrant cells shapes in the dorsolateral epidermis, including dorsoventral length and apical surface area. SJ mutant embryos also show mild defects in actomyosin structures along the leading edge, but laser cutting experiments suggest similar tension and viscoelastic properties in SJ mutant versus wild type epidermis. In a high percentage of SJ mutant embryos, the epidermis tears free from the amnioserosa near the end of DC and live imaging and immunostaining reveal reduced levels of E-cadherin, suggesting that defective adhesion may be responsible for these tears. Supporting this notion, reducing E-cadherin by half significantly enhances the penetrance of DC defects in coracle mutant embryos., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De, Rice, Zulueta-Coarasa, Fernandez-Gonzalez and Ward.)