Perfusion System for Modification of Luminal Contents of Human Intestinal Organoids and Realtime Imaging Analysis of Microbial Populations

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.

First Report of Fruit Rot Caused by Fusarium luffae in Muskmelon in China

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. However, many pathogens can cause decay of muskmelon fruit, including Fusarium spp.. Fusarium spp. are the most important pathogen, affecting muskmelon fruit yield and quality (Wang et al. 2011). In August 2020, fruit rot symptoms were observed on ripening muskmelons (cv. Tianbao) in several fields in Jiyang District, Jinan City of Shandong Province, China. The incidences of infected muskmelon ranged from 15% to 30% and caused an average 20% yield loss. Symptoms appeared as pale brown, water-soaked lesions that were irregular in shape, with the lesion sizes ranging from a small spot (1 to 2 cm) to decay of the entire fruit. The core and surface of infected fruit were colonized and covered with white mycelia. Two infected muskmelons were collected from two fields, 3.5 km apart. Tissues removed from inside the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25°C in the dark for 5 days. Four purified cultures were obtained using the single spore method. On carnation leaf agar (CLA), 3 to 5 septate, falcate, with a pronounced dorsiventral curvature macroconidia with tapered apical cell, and foot-shaped basal cell, measuring 20 to 40 × 3.5 to 4.5 μm. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with the description of F. luffae (Wang et al., 2019). Because these isolates had similar morphology, two representative isolates (XP11 and XP12) were selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolates using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (ITS: MW391509 and MW391510, CAM: MW392789 and MW392790, RPB2: MW392797 and MW392798, TEF1: MW392793 and MW392794). Alignments of a combined dataset of ITS, CAM, RPB2 and TEF1 were made using MAFFT v. 7, and phylogenetic analyses were conducted in MEGA v. 7.0 using the maximum likelihood method. The muskmelon isolates (XP11 and XP12) clustered together with the F. luffae reference strain LC12167 (99% bootstrap). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25°C, the interior of the inoculated muskmelons begun to rot, and the rot lesion expanded from the core towards the surface of the fruit, then white mycelia were produced on the surface. Ten isolations were re-isolated from the infected tissues and confirmed to fulfill Koch’s postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of fruit rot caused by F. luffae in muskmelon in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.

First report of root rot of Schisandra chinensis caused by Fusarium acuminatum in Northeast China

Wuweizi [Schisandra chinensis（Turcz.）Baill.] is used for traditional medicine in northeastern China. In August of 2019, root rot of S. chinensis with an incidence of 30%-50% was observed in a commercial field located in Liaozhong city (41º29’57” N, 122º52’33” E) in the Liaoning province of China. The diseased plants were less vigorous, stunted, and had leaves that turned yellow to brown. Eventually, the whole plant wilted and died. The diseased roots were poorly developed with brown lesion and eventually they would rot. To determine the causal agent, symptomatic roots were collected, small pieces of root with typical lesions were surface sterilized in 2% NaOCl for 3 min, rinsed three times in distilled water, and then plated onto PDA medium. After incubation at 26°C for 5 days, whitish-pink or carmine to rose red colonies on PDA were transferred to carnation leaf agar (CLA). Single spores were isolated with an inoculation needle using a stereomicroscope. Five single conidia isolates obtained from the colonies were incubated at 26°C for 7 days, abundant macroconidia were formed in sporodochia. Macroconidia were falcate, slender, with a distinct curve to the latter half of the apical cell, mostly 3 to 5 septate, measuring 31.3 to 47.8 × 4.8 to 7.5µm (n=50). Microconidia were oval and irregular ovals, 0-1 septate, measuring 5.0 to 17.5 × 2.5 to 17.5µm (n=50). Chlamydospores formed in chains on within or on top of the mycelium. Morphological characteristics of the isolates were in agreement with Fusarium acuminatum (Leslie and Summerell, 2006). To confirm the identity, the partial sequence of the translation elongation factor 1 alpha (TEF1-á) gene of five isolates was amplified using the primers EF-1(ATGGGTAAGGARGACAAG) and EF-2 (GGARGTACCAGTSATCATGTT) (O’Donnell et al. 2015 ) and sequenced. The rDNA internal transcribed spacer (ITS) region for the five isolates was also amplified using the primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTATTGATATGC) (White et al.1990) and sequenced. The identical sequences were obtained, and one representative sequence of isolate WW31-5 was submitted to GenBank. BLASTn analysis of the TEF-á sequence (MW423624) and ITS sequence (MZ145386), revealed 100%（708/685bp, 563/563bp）sequence identity to F. acuminatum MH595498 and MW560481, respectively. Pathogenicity tests were conducted in greenhouse. Inoculums of F. acuminatum was prepared from the culture of WW31-5 incubated in 2% mung beans juice on a shaker (140 rpm) at 26°C for 5 days. Ten roots of 2-years old plants of S. chinensis were immersed in the conidial suspension (2 × 105 conidia/ml) for 6 hours, and another ten roots immersed in sterilized distilled water in plastic bucket for 6 hours. All these plants were planted into pots with sterilized field soil (two plants per pot). Five pots planted with inoculated plants and another five pots planted with uninoculated plants served as controls. All ten pots were maintained in a greenhouse at 22-26°C for 21 days and irrigated with sterilized water. The leaves of the inoculated plants became yellow，gradually dried up, eventually finally all the aboveground parts died. The roots of the inoculated plants were rotted. Non-inoculated control plants had no symptoms. F. acuminatum was reisolated from the roots of inoculated plants and had morphology identical to the original isolate. The experiment was repeated twice with similar results. F. acuminatum has been reported as a pathogen caused root rot of ginseng (Wang et al. 2016) and not reported on Wuweizi in China. To our knowledge, this is the first report of root rot of S. chinensis caused by F. acuminatum. We have also observed the disease at Benxi city of Liaoning Province in 2020 and it has become an important disease in production of S. chinensis and the effective control method should be adopted to reduce losses.

Neopestalotiopsis rosae Causing Black Spot on Leaf and Fruit of Pecan (Carya illinoinensis) in China

Pecan (Carya illinoinensis K. Koch) is very productive and brings good economic returns for mountain dwellers. In recent years, symptoms were observed on both leaves and fruits of pecan in orchards, Fuyang, Anhui Province, and Jiande, Zhejiang Province. On the leaf, spots, rust-colored, long shuttle to irregular shaped, and 1–3 mm in size, firstly appeared between veins. The color of area around the spot gradually changed from green to yellow making a yellow halo. Then, some spots often merged in to a big lesion. On the fruit, symptoms firstly appear as irregular and black spots. The spots gradually spread to most part of the fruit and the core of the fruit turned black at the late stage of pathogenesis. Normally, the leaf infection rate was less than 10%, whereas infection rate of fruit could reach 5–15% which leads to drastic quality reduction and significant yield losses. To isolate the pathogens, fresh infected tissues were cut from samples, disinfected and cultured at 2% water agar at 28 oC till colony could be spotted. Fungal isolates were obtained by mycelia tip transferring. Similar cultural and morphological characters showed on 39 of the total 45 isolates. The mycelia grew rapidly in PDA plates, and colony diameter could reach 8.0 cm after culture at 25 oC in dark for 5 days. Mycelia on surface of the colony were cotton-like and showed obvious growth rings, and turn yellow from the middle on aged cultures. Pycnidia, brown to dark in color, began to form on PDA after 15 days’ culture in the dark. Conidia were fusoid, straight to slightly curved, 4-septate, 23.5–26.3×7.6–9.0μm, average 24.9×8.3μm (n=60) in size. The basal cell was hyaline, conic with a truncate base and thin-walled, average 4.4–5.1μm long (n=60). The three median cells were doliiform and brown with darker septa. The apical cell was hyaline, cylindrical, with 2–3 tubular apical appendages arising from the apical crest, unbranched, filiform, average 21.7–29.2 μm(n=60) long. The cultural and morphological characters of the strains matched the previous descriptions of Neopestalotiopsis sp. (Maharachchikumbura et al., 2014). Their 3 partial genes, ITS, β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1-α), were amplified and sequenced with primers (Lee et al., 2006; Maharachchikumbura et al., 2014). These sequences were submitted in GenBank with accession number of MW504791 (ITS), MW633956 (TEF1-α), and MW633958 (TUB2), seperately. The BLAST results against the GenBank database showed more than 99% identity with sequences claimed from Neopestalotiopsis sp. The multilocus phylogenetic analysis of the three genes showed the isolate was closest with N. rosae. The strains were finally identified as N. rosae by morphological and molecular analysis. Pathogenicity testing was performed by spreading 20μl conidia suspension drop (1 × 106 conidia ml-1) on surface of wounded or unwounded leaves and fruits. The pecan variety MaHan was used for inoculation. Wounds were made by scratching with sterile needles, and then a drop of 20μl conidia suspension was spread on the wounded area. For unwounded inoculation, a drop of 20μl conidia suspension was spread without wound procedure. The same amount of water drops were spread on the surface of wounded and unwounded tissues as controls. The plant parts were wrapped with PE films to keep moisture after drop spreading. Ten trees were selected for inoculation, and 10 leaves and fruits were randomly selected separately for wounded and unwounded inoculation and corresponding controls in each tree. The similar symptoms appeared from 3 dpi on leaves and 9 dpi on fruits, and finally showed on more than 90% wounded inoculated leaves or fruits and on less than 50% after unwounded inoculation, while controls remained symptomless. N. rosae was consistently re-isolated from inoculated parts with symptoms, while not from the control. Therefore, N. rosae was confirmed as the pathogen causing black spot on leaf and fruit of pecan (C. illinoinensis). To the best of our knowledge, this is the first report of N. rosae causing black spot disease of pecan (C. illinoinensis). In recent years, both the infection area and severity of this disease grow rapidly, which means the potential of the disease to become a big problem in local orchards.

First Report of Fusarium pernambucanum Causing Fruit Rot of Muskmelon in China

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. However, many pathogens can cause decay of muskmelons; among them, Fusarium spp. is the most important pathogen, affecting fruit yield and quality (Wang et al. 2011). In May 2017, fruit rot symptoms were observed on ripening muskmelons (cv. Jipin Zaoxue) in several fields in Liaocheng of Shandong Province, China. Symptoms appeared as brown, water-soaked lesions, irregularly circular in shape, with the lesion size ranging from a small spot (1 to 2 cm) to the decay of the entire fruit. The core and the surface of the infected fruit were covered with white to rose-reddish mycelium. Two infected muskmelons were collected from each of two fields, 10 km apart. Tissues from the inside of the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25 °C in the dark for 5 days. Four purified cultures were obtained using the single spore method. On carnation leaf agar (CLA), macroconidia had a pronounced dorsiventral curvature, falcate, 3 to 5 septa, with tapered apical cell, and foot-shaped basal cell, measuring 19 to 36 × 4 to 6 μm. Chlamydospores were abundant, 5.5–7.5 μm wide, and 5.5–10.5 μm long, ellipsoidal or subglobose. No microconidia were observed. These morphological characteristics were consistent with the descriptions of F. pernambucanum (Santos et al. 2019). Because these isolates had similar morphology, one representative isolate was selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolate using the CTAB method. The nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), translation elongation factor 1-α gene (TEF1), RNA polymerase II second largest subunit gene (RPB2), calmodulin (CAM) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (MN822926, MN856619, MN856620, and MN865126). Based on the combined dataset of ITS, TEF1, RPB2, CAM, alignments were made using MAFFT v. 7, and phylogenetic analyses were processed in MEGA v. 7.0 using the maximum likelihood method. The studied isolate (XP1) clustered together with F. pernambucanum reference strain URM 7559 (99% bootstrap). To perform pathogenicity test, 10 μl of spore suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25 °C, the interior of the inoculated muskmelons begun to rot, and the rot lesion was expanded from the core towards the surface of the fruit, then white mycelium produced on the surface. The same fungus was re-isolated from the infected tissues and confirmed to fulfill the Koch’s postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of F. pernambucanum causing of fruit rot of muskmelon in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.

Mosses: Accessible Systems for Plant Development Studies

Mosses are a cosmopolitan group of land plants, sister to vascular plants, with a high potential for molecular and cell biological research. The species Physcomitrium patens has helped gaining better understanding of the biological processes of the plant cell, and it has become a central system to understand water-to-land plant transition through 2D-to-3D growth transition, regulation of asymmetric cell division, shoot apical cell establishment and maintenance, phyllotaxis and regeneration. P. patens was the first fully sequenced moss in 2008, with the latest annotated release in 2018. It has been shown that many gene functions and networks are conserved in mosses when compared to angiosperms. Importantly, this model organism has a simplified and accessible body structure that facilitates close tracking in time and space with the support of live cell imaging set-ups and multiple reporter lines. This has become possible thanks to its fully established molecular toolkit, with highly efficient PEG-assisted, CRISPR/Cas9 and RNAi transformation and silencing protocols, among others. Here we provide examples on how mosses exhibit advantages over vascular plants to study several processes and their future potential to answer some other outstanding questions in plant cell biology.

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing

Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

First Report of Fusarium culmorum Causing Maize Stalk Rot in China

Maize stalk rot has become one of the most important diseases in maize production in China. From 2017 to 2019, a survey was conducted to determine the population diversity of Fusarium species associated with maize diseases in 18 cities across Henan Province. Maize stalk rot with an incidence of more than 20% that caused yield losses up to 30% was observed on maize variety Zhengdan958, which was grown in two continuous maize fields in Zhumadian City, Henan Province. The stem tissues from the boundary between diseased and healthy pith were chopped into small pieces (3 × 8 mm), disinfected (70% ethanol for 1 min) and then placed onto potato dextrose agar (PDA) amended with L-(+)-Lactic-acid (1 g/L) and incubated at 25°C for 4 days. Colonies on PDA produced fluffy, light yellow aerial mycelium and purple to deep brick red pigment at 25°C (Fig 1A, 1B). On carnation leaf agar (CLA), macroconidia in orange sporodochia formed abundantly, but microconidia were absent. Macroconidia were short and thick-walled, had 3 to 5 septa, a poorly developed foot cell and rounded apical cell (Fig 1C). These characteristics matched the description of Fusarium culmorum (Leslie and Summerell 2006) and isolates DMA268-1-2 and HNZMD-12-7 were selected for further identity confirmation. Species identification was confirmed by partial sequences of three phylogenic loci (EF1-α, RPB1, and RPB2) using the primer pairs EF1/EF2, CULR1F/CULR1R, and CULR2F/CULR2R, respectively (O'Donnell et al., 1998). The consensus sequences from the two isolates were deposited in GenBank (MZ265416 and MZ265417 for TEF, respectively; MZ265412 and MZ265414 for RPB1, respectively; MZ265413 and MZ265415 for RPB2). BLASTn searches indicated that the nucleotide sequences of the three loci of the two isolates revealed 99% to 100% similarity to those of F. culmorum strains deposited in the GenBank, Fusarium-ID, and MLST databases (Supplementary Table 1~3). Pathogenicity test was conducted at the flowering-stage using Zhengdan958 and Xundan20 plants according to previously described method (Zhang et al., 2016; Cao et al., 2021; Zhang et al., 2021). The second or third internodes of thirty flowering plants were drilled to make a wound approximately 8 mm in diameter using an electric drill. Approximately 0.5 mL inoculum (125 mL colonized PDA homogenized with 75 mL sterilized distilled water) was injected into the wound and sealed with Vaseline and Parafilm to maintain moisture and avoid contamination. Sterile PDA slurry was used as a control. Thirty days after inoculation, the dark-brown, soft rot of pith tissues above and below the injection sites were observed, and some plants were severely rotten and lodged (Fig 1D, 1E). These symptoms were similar to those observed in the field. No symptoms were observed on control plants. The same pathogen was re-isolated from the inoculated stalk lesions but not from the control, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of F. culmorum as the causal agent of stalk rot on maize plants in China. Also, this fungus has been reported to cause maize ear rot in China (Duan et al. 2016) and produce mycotoxins such as trichothecenes, nivalenol, and zearalenone that cause toxicosis in animals (Leslie and Summerell 2006). The occurrence of maize stalk rot and ear rot caused by F. culmorum should be monitored due to the potential risk for crop loss and mycotoxin contamination.

First report of Fusarium cf. longipes associated with maize stalk rot in China

In a field survey from 2017 to 2019, Fusarium stalk rot symptoms including discolored, disintegrated stalk pith tissues and lodged plants were observed in maize hybrid lines Fuyu1611, Jidan66, and Danyu8439 grown in fields in Anshan (40o49′39′′N, 122 o34′6′′E), Liaoning province. Its incidence ranged from 15% to 20% and caused a yield loss of up to 30%. Infected pieces of stem tissues were dissected and then sterilized with 1% NaOCl for 1 min, 70% ethanol for 1 min, rinsed 3 times with sterilized ddH2O, and dried with filter paper in hood. Three pieces were placed onto Potato dextrose agar (PDA) and incubated at 25 °C for 5 days. The colonies were single-spore subcultured on PDA at 25 °C for 2 weeks (Leslie and Summerell 2006). Morphological features were observed on PDA and carnation leaf agar (CLA). The average mycelial growth rate was 4.5 to 10.3 mm/day at 25 °C on PDA. The colonies produced aerial mycelia, varying from dense white to grayish-rose, and secreted red pigments in the agar (Fig. 1A; 1B). Macroconidia produced on CLA were long and relatively slender, commonly 4- to 7-septate, averaging 85.6 × 5.2 μm, with thick walls and pronounced dorsiventral curvature with a distinctly foot-shaped and elongated basal cell and an apical cell that was whip-like (Fig. 1C). Microconidia were rarely observed on PDA or CLA. Morphological characteristics of the isolates were similar to the features of Fusarium longipes as previously described (Leslie and Summerell 2006). The portions of three phylogenic loci (EF1-α, RPB1, RPB2) were PCR amplified using the primer pairs EF1/EF2 (O'Donnell et al, 1998), lonR1F/lonR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3 and 5-CCAATGGACTGGGCAGCCAAAACGCC-3) and lonR2F/lonR2R (5-TATACATTTGCCTCCACTCTTTCCCAT-3 and 5-CGGAGCTTGCGTCCGGTGTGGCCGTTG-3) and sequenced. The consensus sequences were submitted to GenBank (MT513215 and MT997083 for TEF, MT513213 and MT997088 for RPB1; MT513214 and MW020572 for RPB2). BLASTn searches indicated that the nucleotide sequences of the three loci of the two isolates shared 94.52% to 99.69% identity with sequences of F. longipes strains deposited in the GenBank, Fusarium-ID and Fusarium MLST databases (Supplementary Table 1, 3, 4). A phylogram inferred via maximum likelihood analysis of the combined EF-1α, RPB1, RPB2 partial sequence data of Fusarium species (Supplementary Table 2) was inferred using the CIPRIES website (https://www.phylo.org). Isolates LNAS-05-A and LNAS-09-A clustered with F. longipes, with 98% bootstrap support (Fig. 2). Pathogenicity tests were conducted on three-leaf-stage seedlings and flowering-stage c.v. Zhengdan958 and B104 plants according to previously described methods (Ye et al., 2013; Zhang et al. 2016) with minor modifications. Three days after the roots of the seedlings were inoculated with 1 × 106 macroconidia solution, the leaves and stems exhibited typical wilt symptoms (Fig. 1D). Twenty flowering-stage maize plants were drilled individually at the second or third node above the soil using an electric drill (Bosch TSR1080-2-Li) to create a hole (8 mm in diameter). An approximately 0.5 mL mycelia plug (125 mL homogenized hyphal mats + 75 mL sterilized ddH2O) was injected into the hole and covered with Vaseline. Sterilized PDA plugs were used as a control. The stalk tissue of the split internodes turned dark brown and the brown area expanded above and below the injection site by 14 dpi. All of the inoculated plants developed characteristic stalk rot symptoms, whereas no symptoms were observed in the controls (Fig. 1E). The pathogen was re-isolated, and its identity was confirmed by sequencing the above mentioned loci. F. longipes was generally regarded as a tropical Fusarium species (Leslie and Summerell 2006). This is the first report that F. cf. longipes can cause stalk rot of maize under filed condition in a temperate, typical corn belt region of China.

Induction of apically mistrafficked epiregulin disrupts epithelial polarity via aberrant EGFR signaling

ABSTRACT In polarized MDCK cells, disruption of the tyrosine-based YXXΦ basolateral trafficking motif (Y156A) in the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG), results in its apical mistrafficking and transformation in vivo. However, the mechanisms underlying these dramatic effects are unknown. Using a doxycycline-inducible system in 3D Matrigel cultures, we now show that induction of Y156A EREG in fully formed MDCK cysts results in direct and complete delivery of mutant EREG to the apical cell surface. Within 3 days of induction, ectopic lumens were detected in mutant, but not wild-type, EREG-expressing cysts. Of note, these structures resembled histological features found in subcutaneous xenografts of mutant EREG-expressing MDCK cells. These ectopic lumens formed de novo rather than budding from the central lumen and depended on metalloprotease-mediated cleavage of EREG and subsequent EGFR activity. Moreover, the most frequent EREG mutation in human cancer (R147stop) resulted in its apical mistrafficking in engineered MDCK cells. Thus, induction of EREG apical mistrafficking is sufficient to disrupt selective aspects of polarity of a preformed polarized epithelium. This article has an associated First Person interview with the first author of the paper.