Septate Junctions Research Articles

Paracellular barriers: Advances in assessing their contribution to renal epithelial function

Regulation of salt and water balance occupies a dominant role in the physiology of many animals and often relies on the function of the renal system. In the mammalian kidney, epithelial ion and water transport requires high degree of coordination between the transcellular and paracellular pathways, the latter being defined by the intercellular tight junctions (TJs). TJs seal the paracellular pathway in a highly specialized manner, either by forming a barrier against the passage of solutes and/or water or by allowing the passage of ions and/or water through them. This functional TJ plasticity is now known to be provided by the members of the claudin family of tetraspan proteins. Unlike mammalian nephron, the renal structures of insects, the Malpighian tubules, lack TJs and instead have smooth septate junctions (sSJs) as paracellular barrier forming junctions. Many questions regarding the molecular and functional properties of sSJs remain open but research on model species have begun to inform our understanding. The goal of this commentary is to highlight key concepts and most recent findings that have emerged from the molecular and functional dissection of paracellular barriers in the mammalian and insect renal epithelia.

Sinuous Is a Claudin Required for Locust Molt in Locusta migratoria.

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.

The Drosophila blood–brain barrier invades the nervous system in a GPCR-dependent manner

The blood–brain barrier (BBB) represents a crucial interface between the circulatory system and the brain. In Drosophila melanogaster, the BBB is composed of perineurial and subperineurial glial cells. The perineurial glial cells are small mitotically active cells forming the outermost layer of the nervous system and are engaged in nutrient uptake. The subperineurial glial cells form occluding septate junctions to prevent paracellular diffusion of macromolecules into the nervous system. To address whether the subperineurial glia just form a simple barrier or whether they establish specific contacts with both the perineurial glial cells and inner central nervous system (CNS) cells, we undertook a detailed morphological analysis. Using genetically encoded markers alongside with high-resolution laser scanning confocal microscopy and transmission electron microscopy, we identified thin cell processes extending into the perineurial layer and into the CNS cortex. Interestingly, long cell processes were observed reaching the glia ensheathing the neuropil of the central brain. GFP reconstitution experiments highlighted multiple regions of membrane contacts between subperineurial and ensheathing glia. Furthermore, we identify the G-protein-coupled receptor (GPCR) Moody as negative regulator of the growth of subperineurial cell processes. Loss of moody triggered a massive overgrowth of subperineurial cell processes into the CNS cortex and, moreover, affected the polarized localization of the xenobiotic transporter Mdr65. Finally, we found that GPCR signaling, but not septate junction formation, is responsible for controlling membrane overgrowth. Our findings support the notion that the Drosophila BBB is able to bridge the communication gap between circulation and synaptic regions of the brain by long cell processes.

The polarity protein Yurt associates with the plasma membrane via basic and hydrophobic motifs embedded in its FERM domain.

The subcellular distribution of the polarity protein Yurt (Yrt) is subjected to a spatio-temporal regulation in Drosophila melanogaster embryonic epithelia. After cellularization, Yrt binds to the lateral membrane of ectodermal cells and maintains this localization throughout embryogenesis. During terminal differentiation of the epidermis, Yrt accumulates at septate junctions and is also recruited to the apical domain. Although the mechanisms through which Yrt associates with septate junctions and the apical domain have been deciphered, how Yrt binds to the lateral membrane remains as an outstanding puzzle. Here, we show that the FERM domain of Yrt is necessary and sufficient for membrane localization. Our data also establish that the FERM domain of Yrt directly binds negatively charged phospholipids. Moreover, we demonstrate that positively charged amino acid motifs embedded within the FERM domain mediates Yrt membrane association. Finally, we provide evidence suggesting that Yrt membrane association is functionally important. Overall, our study highlights the molecular basis of how Yrt associates with the lateral membrane during the developmental time window where it is required for segregation of lateral and apical domains.

Damage control of epithelial barrier function in dynamic environments

Epithelial tissues cover the surfaces and lumens of the internal organs of multicellular animals and crucially contribute to internal environment homeostasis by delineating distinct compartments within the body. This vital role is known as epithelial barrier function. Epithelial cells are arranged like cobblestones and intricately bind together to form an epithelial sheet that upholds this barrier function. Central to the restriction of solute and fluid diffusion through intercellular spaces are occluding junctions, tight junctions in vertebrates and septate junctions in invertebrates. As part of epithelial tissues, cells undergo constant renewal, with older cells being replaced by new ones. Simultaneously, the epithelial tissue undergoes relative rearrangement, elongating, and shifting directionally as a whole. The movement or shape changes within the epithelial sheet necessitate significant deformation and reconnection of occluding junctions. Recent advancements have shed light on the intricate mechanisms through which epithelial cells sustain their barrier function in dynamic environments. This review aims to introduce these noteworthy findings and discuss some of the questions that remain unanswered.

ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism detecting alteration of septate junction integrity in Drosophila epithelial cells.

Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of barrier functions to allow tissue integrity maintenance is poorly characterised. Cellular junctions play an important role in this process and intracellular traffic contribute to their homeostasis. Here, we reveal that, in Drosophila pupal notum, alteration of the bi- or tricellular septate junctions (SJs) triggers a mechanism with two prominent outcomes. On one hand, there is an increase in the levels of E-cadherin, F-actin, and non-muscle myosin II in the plane of adherens junctions. On the other hand, β-integrin/Vinculin-positive cell contacts are reinforced along the lateral and basal membranes. We found that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution, reduces degradation and instead favours recycling of SJ components, an effect that extends to other recycled transmembrane protein cargoes including Crumbs, its effector β-Heavy Spectrin Karst, and β-integrin. We propose a mechanism by which epithelial cells, upon sensing alterations of the SJ, reroute the function of Shrub to adjust the balance of degradation/recycling of junctional cargoes and thereby compensate for barrier junction defects to maintain epithelial integrity.

Comparative analysis of the cytoarchitecture of the excretory bladder of adult Digenea (Platyhelminthes) with consideration of the presence of mineralized excretory corpuscles in marine and freshwater adult worms

The ultrastructural differences are shown between the cytoarchitecture of the excretory bladder and excretory inclusions in four digenean species, two azygiids, the marine Otodistomum cestoides and the freshwater Azygia lucii, the marine derogenid Derogenes varicus and the freshwater allocreadiid Acrolichanus auriculatus. The unusual cytoarchitecture of the bladder epithelium of the azygiid digeneans, consisting along its entire length of two alternating, morphologically different zones, tegumental and cellular excretory epithelial zones, connected by septate junctions, has recorded for the first time for the Digenea and, in general, for the Neodermata. It, possible, suggests the participation of the tegumental distal cytoplasmic layer in the formation of their excretory bladder epithelium. Like most digeneans, the excretory bladder of A. auriculatus and D. varicus has a syncytial epithelial lining. Based on available literature and our own results, we can confirm the presence of the excretory corpuscles in adult marine digeneans and their absence from freshwater species, regardless of the digenean localization in their host. The present study shown that in marine digeneans, the excretory corpuscles are associated with specialized excretory cells or excretory syncytial epithelium. Ultrastructural data were obtained on the possible growth of the excretory bladder epithelium due to the migration of undifferentiated cells into the epithelial lining in studied marine species. We may assume that the bladder epithelium of marine adult digeneans specializes, in addition to the excretory function, in osmoregulatory function.

Compromised junctional integrity phenocopies age-dependent renal dysfunction in Drosophila Snakeskin mutants.

Transporting epithelia provide a protective barrier against pathogenic insults while allowing the controlled exchange of ions, solutes and water with the external environment. In invertebrates, these functions depend on formation and maintenance of 'tight' septate junctions (SJs). However, the mechanism by which SJs affect transport competence and tissue homeostasis, and how these are modulated by ageing, remain incompletely understood. Here, we demonstrate that the Drosophila renal (Malpighian) tubules undergo an age-dependent decline in secretory capacity, which correlates with mislocalisation of SJ proteins and progressive degeneration in cellular morphology and tissue homeostasis. Acute loss of the SJ protein Snakeskin in adult tubules induced progressive changes in cellular and tissue architecture, including altered expression and localisation of junctional proteins with concomitant loss of cell polarity and barrier integrity, demonstrating that compromised junctional integrity is sufficient to replicate these ageing-related phenotypes. Taken together, our work demonstrates a crucial link between epithelial barrier integrity, tubule transport competence, renal homeostasis and organismal viability, as well as providing novel insights into the mechanisms underpinning ageing and renal disease.

The neuro-exocrine secretion: A new type of gland in tapeworms?

The phenomenon of exocrine secretion via nervous cells into the host tissue has been discovered in cestodes. In five cestode species of different orders specialized “cup-shaped” free nerve endings located in the tegument have been found. Their ultrastructure is characterized by the presence of a septate junction, a thin support ring and neurosecretory vesicles 90–110 nm in diameter, which are secreted onto the surface of the tegument through a thin pore. The phenomenon is referred to in this article as the neuro-exocrine secretion. We observed a direct relationship between neurosecretory processes in the deep subtegument and free endings in a series of ultrathin sections in two species. The peripheral neurosecretory neurons of species studied are characterized by similar ultrastructural features: size and location; diameter of neurosecretory granules; absence of microtubules and mitochondria in the neurites. The size of neurosecretory granules has been found to decrease from perikaryon towards neurosecretory terminals that lead to the tegument. In two species, we examined the neurosecretion during incubation in the host's blood serum. Depending on the time of incubation we have shown the changes a) in the diameter of the cup-shaped endings, b) in the number of secretory vesicles in the endings; c) changes in number and diameter of neurosecretory vesicles in the processes of neurosecretory neurons in the subtegument. The detected changes differ in D.dendriticus and L.interrupta and, taken together, indirectly confirm the secretory specialization of the cup-shaped endings. Supposed targets for the neurosecretory neurons in the studied cestodes are the following: (a) eccrine frontal gland ducts, especially their terminal regions involved in the release of secretory products; (b) longitudinal and circular muscles in the subtegument region; (c) the basal membrane of the tegument. Besides the discovered secretion vesicles through the cup-shaped terminals, we observed vacuoles derived from the basal membrane of the tegument containing extracellular substances released into the host tissue. Their possible role in the release of neurosecretory substances is discussed. Considering the data acquired via immunocytochemical methods, an assumption about involvement of FMRFamide-like related peptides (FaRPs) in the neuro-exocrine secretion is proposed. Possible functions of the neuro-exocrine secretion are discussed in the context of host-parasite interactions.

The Lipid Phosphate Phosphatase Wunen Promotes Eggshell Formation and Is Essential for Fertility in Drosophila.

The eggshell that surrounds insect eggs acts as a barrier, protecting against biotic factors and desiccation. The eggshell is a multi-layered structure which is synthesised by the somatic follicle cells that surround the developing oocyte. Although the temporal order of expression of the protein eggshell components goes someway to explaining how the different layers are built up, but how the precise three-dimensional structure is achieved and how lipid components responsible for desiccation resistance are incorporated are poorly understood. In this paper, we demonstrate that wunen, which encodes a lipid phosphate phosphatase, is necessary for fertility in Drosophila females. Compared to sibling controls, females null for wunen lay fewer eggs which subsequently collapse such that no larvae emerge. We show that this is due to a requirement for wunen in the ovarian follicle cells which is needed to produce an ordered and functional eggshell. Knockdown of a septate junction component also results in collapsed eggs, supporting the idea that similar to its role in embryonic tracheal development, Wunen in follicle cells also promotes septate junction function.

Requirement of Snakeskin for normal functions of midgut and Malpighian tubules in Henosepilachna vigintioctopunctata.

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.

Microbiome and electron microscopy analyses of the mechanisms underlying the effects of Bacillus thuringiensis on Dioryctria abietella

The ability of the bacteria Bacillus thuringiensis (Bt) to effectively kill target insects is well-established. However, the mechanisms by which Bt interacts with gut microbes to trigger insect cell death is in forest pests remain unclear. Dioryctria abietella is one of the most destructive forest pests for coniferous trees. In the present work, D. abietella was used as a model to investigate the effects of Bacillus thuringiensis subsp. galleriae 05041 strain (Bt05041, 1 × 108 CFU mL−1) on the gut microbiota and midgut cells at different time points after treatment (3, 6, 9, 12, and 24 h). Treatment with Bt05041 significantly enriched the gut bacterial community, altered the composition of the gut microbiota, and resulted in the dysfunction of gut cells. Bt05041 treatment significantly increased the OTUs (operational taxonomic units) compared to the number in the control group. The genera Enterococcus, Wolbachia, Bacillus, and unclassified Enterobacteriaceae had relatively high abundances after Bt05041 treatment. The midgut cells of D. abietella deteriorated significantly in response to Bt05041, as determined by transmission electron microscopy. Changes in cell morphology included the disruption of microvilli, deformation of organelles, and cytoplasmic vacuolation. With the extension of the Bt05041 treatment duration, the basal plasma membrane and septate junction gradually ruptured until cells were completely dissolved. In conclusion, our findings reveal the effects of Bt05041 exposure for various durations on gut microbes and cells, clarify the detailed mechanism underlying Bt-mediated apoptosis, and provide new insights into the use of Bt for forest pest control.

The septate junction component bark beetle is required for Drosophila intestinal barrier function and homeostasis

Age-related loss of intestinal barrier function has been documented across species, but the causes remain unknown. The intestinal barrier is maintained by tight junctions (TJs) in mammals and septate junctions (SJs) in insects. Specialized TJs/SJs, called tricellular junctions (TCJs), are located at the nexus of three adjacent cells, and we have shown that aging results in changes to TCJs in intestines ofadult Drosophila melanogaster. We now demonstrate that localization of theTCJ protein bark beetle (Bark) decreases in aged flies. Depletion of bark fromenterocytes in young flies led to hallmarks of intestinal aging and shortened lifespan, whereas depletion of bark in progenitor cells reduced Notch activity, biasing differentiation toward the secretory lineage. Our data implicate Bark in EC maturation and maintenance of intestinal barrier integrity. Understanding the assembly and maintenance of TCJs to ensure barrier integrity may lead to strategies to improve tissue integrity when function is compromised.

Cell-intrinsic and -extrinsic functions of the ESCRT-III component shrub in cytokinetic abscission of Drosophila sensory organ precursor.

Although the molecular mechanisms governing abscission of isolated cells are largely decrypted, those of epithelial progenitors surrounded by epidermal cells (ECs) connected via cellular junctions remain largely unexplored. Here, we investigated the remodeling of the paracellular diffusion barrier ensured by septate junctions (SJs) during cytokinesis of Drosophila sensory organ precursor (SOP). We report that SOP cytokinesis involves the coordinated, polarized assembly and remodeling of SJs in the dividing cell and its neighbors, which remain connected to the former via membrane protrusions pointing towards the SOP midbody. SJs assembly and midbody basal displacement occur faster in SOPs than in ECs, leading to a quicker disentanglement of neighboring cells membrane protrusions prior to midbody release. As reported in isolated cells, the endosomal sorting complex required for transport-III component Shrub/CHMP4B is recruited at the midbody and cell-autonomously regulates abscission. In addition, Shrub is recruited to membrane protrusions, is required for SJ integrity, and alteration of SJ integrity leads to premature abscission. Our study uncovers cell- intrinsic and -extrinsic functions of Shrub in coordinating remodeling of the SJs and SOP abscission.

Coordination of tissue homeostasis and growth by the Scribble-α-Catenin-Septate junction complex

Maintaining apicobasal polarity (ABP) is crucial for epithelial integrity and homeostasis during tissue development. Although intracellular mechanisms underlying ABP establishment have been well studied, it remains to be addressed how theABP coordinates tissue growth and homeostasis. By studying Scribble, a key ABP determinant, we address molecular mechanisms underlying ABP-mediated growth control in the Drosophila wing imaginal disc. Our data reveal that genetic and physical interactions between Scribble, Septate junction complex and α-Catenin appear to be key for sustaining ABP-mediated growth control. Cells with conditional scribble knockdown instigate the loss of α-Catenin, ultimately leading to the formation of neoplasia accompanying with activation of Yorkie. In contrast, cells expressing wild type scribble progressively restore ABP in scribble hypomorphic mutant cells in a non-autonomous manner. Our findings provide unique insights into cellular communication among optimal and sub-optimal cells to regulate epithelial homeostasis and growth.

Identification of the genes encoding candidate septate junction components expressed during early development of the sea urchin, Strongylocentrotus purpuratus, and evidence of a role for Mesh in the formation of the gut barrier

Septate junctions (SJs) evolved as cell-cell junctions that regulate the paracellular barrier and integrity of epithelia in invertebrates. Multiple morphological variants of SJs exist specific to different epithelia and/or phyla but the biological significance of varied SJ morphology is unclear because the knowledge of the SJ associated proteins and their functions in non-insect invertebrates remains largely unknown. Here we report cell-specific expression of nine candidate SJ genes in the early life stages of the sea urchin Strongylocentrotus purpuratus. By use of in situ RNA hybridization and single cell RNA-seq we found that the expression of selected genes encoding putatively SJ associated transmembrane and cytoplasmic scaffold molecules was dynamically regulated during epithelial development in the embryos and larvae with different epithelia expressing different cohorts of SJ genes. We focused a functional analysis on SpMesh, a homolog of the Drosophila smooth SJ component Mesh, which was highly enriched in the endodermal epithelium of the mid- and hindgut. Functional perturbation of SpMesh by both CRISPR/Cas9 mutagenesis and vivo morpholino-mediated knockdown shows that loss of SpMesh does not disrupt the formation of the gut epithelium during gastrulation. However, loss of SpMesh resulted in a severely reduced gut-paracellular barrier as quantitated by increased permeability to 3–5 ​kDa FITC-dextran. Together, these studies provide a first look at the molecular SJ physiology during the development of a marine organism and suggest a shared role for Mesh-homologous proteins in forming an intestinal barrier in invertebrates. Results have implications for consideration of the traits underlying species-specific sensitivity of marine larvae to climate driven ocean change.

Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium

Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. Herein, by combining live imaging and physical modeling, we showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, we found that M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, we developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions.

Glyphosate-Based Herbicide Causes Cellular Alterations to Gut Epithelium of the Neotropical Stingless Bee Melipona quadrifasciata quadrifasciata (Hymenoptera: Meliponini).

Glyphosate-based herbicides (GBH) are the best-selling pesticides in Brazil, with hundreds of thousands of tons sold per year. There is no study investigating morphological alterations caused by GBH on the epithelium of the gut in bees. Here, we aimed to demonstrate effects of chronic ingestion of GBH in the midgut digestive cells of the Brazilian stingless bee Melipona quadrifasciata quadrifasciata Lepeletier 1836. We kept forager workers of M. quadrifasciata in laboratory conditions and fed on food contaminated with three different concentrations of GBH for 10days, after which the midgut digestive cell structure and ultrastructure were analyzed. The presence of GBH in food did not affect food consumption, indicating that M. quadrifasciata bees do not reject food contaminated with GBH. As digestive cells of the midgut release apocrine secretion as a detoxication mechanism, we expected that the ingestion of food contaminated with GBH in the present study affect the height of midgut digestive cells. However, such reduction did not occur, probably because of the low-test concentrations. Although there were differences in digestive cell ultrastructure, ingestion of GBH impaired midgut digestive cell cohesion by disorganizing the smooth septate junctions between cells, which may probably be caused by the adjuvant "polyethoxylated tallow amine" present in the GBH. Previous studies demonstrated that GBH increase bees' sensibility to intestine infections, based on the present results we hypothesized that the loss of cell cohesion in the midgut epithelium favors pathogenic microbial infections and harms food absorption, increasing bees' mortality.

Knockdown of Na,K-ATPase β-subunits in Oncopeltus fasciatus induces molting problems and alterations in tracheal morphology.

The ubiquitously expressed transmembrane enzyme Na,K-ATPase (NKA) is vital in maintaining functionality of cells. The association of α- and β-subunits is believed to be essential for forming a functional enzyme. In the large milkweed bug Oncopeltus fasciatus four α1-paralogs and four β-subunits exist that can associate into NKA complexes. This diversity raises the question of possible tissue-specific distribution and function. While the α1-subunits are known to modulate cardenolide-resistance and ion-transport efficiency, the functional importance of the β-subunits needed further investigation. We here characterize all four different β-subunits at the cellular, tissue, and whole organismal scales. A knockdown of different β-subunits heavily interferes with molting success resulting in strongly hampered phenotypes. The failure of ecdysis might be related to disrupted septate junction (SJ) formation, also reflected in β2-suppression-induced alteration in tracheal morphology. Our data further suggest the existence of isolated β-subunits forming homomeric or β-heteromeric complexes. This possible standalone and structure-specific distribution of the β-subunits predicts further, yet unknown pump-independent functions. The different effects caused by β knockdowns highlight the importance of the various β-subunits to fulfill tissue-specific requirements.

De novo apical domain formation inside the Drosophila adult midgut epithelium.

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.