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Basal stem cell progeny establish their apical surface in a junctional niche during turnover of an adult barrier epithelium

Nature Cell Biology volume 25, pages 658–671 (2023)Cite this article

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Abstract

Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. New replacement cells—the progeny of basal stem cells—are born without barrier-forming structures such as a specialized apical membrane and occluding junctions. Here, we investigate how new progeny acquire barrier structures as they integrate into the intestinal epithelium of adult Drosophila. We find they gestate their future apical membrane in a sublumenal niche created by a transitional occluding junction that envelops the differentiating cell and enables it to form a deep, microvilli-lined apical pit. The transitional junction seals the pit from the intestinal lumen until differentiation-driven, basal-to-apical remodelling of the niche opens the pit and integrates the now-mature cell into the barrier. By coordinating junctional remodelling with terminal differentiation, stem cell progeny integrate into a functional, adult epithelium without jeopardizing barrier integrity.

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Fig. 1: The apex of a differentiating enteroblast contacts the SJ of its neighbour enterocytes.
Fig. 2: SJ and apical membrane morphology define six stages of barrier integration.
Fig. 3: Synchronized differentiation drives Su(H)-lacZ+ cells through sequential stages of PAC integration.
Fig. 4: PAC integration involves basally directed growth of a transitional SJ with distinct apical EC–EC and basal PC–EC zones.
Fig. 5: FIB-SEM reveals the 3D ultrastructure of early-stage SJs.
Fig. 6: Cells must form SJs and grow to integrate.
Fig. 7: PACs are intercellular, split-polarity lumens that are sealed off from the gut lumen.

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Data availability

EM data are available through EMBL-EBI BioStudies under accession number S-BSST946. Source data are provided with this paper. All other data are available from the corresponding author on reasonable request.

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Acknowledgements

We are grateful to A. Bardin, D. Bilder, N. Buchon, J. de Navascues, M. Furuse, Y. Inoue, H. Jasper, S. Siegrist, N. Tapon and Drosophila stock centres (Bloomington Drosophila Stock Center (NIH P40OD018537), Vienna Drosophila Resource Center (Dietzl et al., 2007), Kyoto Drosophila Genomics and Genetic Resources) for fly stocks; M. Furuse, S. Russell and X. Yang for antibodies; M. Petersen and E. Smith for illustrations; S. Xie for Python support; and J. Mulholland and K. Lee for microscopy support. Confocal microscopy was performed at the Stanford Beckman Cell Sciences Imaging Facility (NIH 1S10OD01058001A1, NIH 1S10OD010580). We thank D. Bryant, T. Reiff, D. St. Johnston, J. Chen and members of the O’Brien lab for invaluable discussions. A. Galenza is supported by a Canadian Institutes of Health Research Fellowship MFE 181906. P.M.R. was supported by a Stanford Bio-X Bowes Graduate Fellowship, an EMBO Short-Term Travelling Fellowship, and a Stanford DARE Graduate Fellowship (Diversifying Academia, Recruiting Excellence). The authors acknowledge the financial support by the Faculty of Biology and Medicine of the University of Lausanne and of the Swiss National Science Foundation, R’Equip Grant 316030_128692. This work was supported by NIH R01GM116000-01A1, NIH R35GM141885-01, NIH 1R01DK128485-01A1 and ACS RSG-17-167-01 to L.E.O. L.E.O. is an investigator of the Chan-Zuckerberg Biohub.

Author information

Author notes

  1. Paola Moreno-Roman

    Present address: Foldscope Instruments, Inc., Palo Alto, CA, USA

  2. Alain Debec

    Present address: Institute of Ecology and Environmental Sciences, iEES, Sorbonne University, UPEC, CNRS, IRD, INRA, Paris, France

  3. Bruno M. Humbel

    Present address: Provost’s Office, Okinawa Institute of Science and Technology, Tancha, Japan

  4. These authors contributed equally: Anthony Galenza, Paola Moreno-Roman.

Authors and Affiliations

  1. Department of Molecular & Cellular Physiology and Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Anthony Galenza, Paola Moreno-Roman, Yu-Han Su, Lehi Acosta-Alvarez & Lucy Erin O’Brien

  2. Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France

    Alain Debec & Antoine Guichet

  3. Luminint Consulting Group, LLC, San Carlos, CA, USA

    Jon-Michael Knapp

  4. Université de Lausanne, Bâtiment Biophore, Quartier Sorge, Lausanne, Switzerland

    Caroline Kizilyaprak, Bruno M. Humbel & Irina Kolotuev

  5. Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Bruno M. Humbel

  6. Chan-Zuckerberg Biohub, San Francisco, CA, USA

    Lucy Erin O’Brien

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Contributions

P.M.-R. and L.E.O. conceived and designed the initial study. A. Galenza and L.E.O. conceived and designed the revised study. P.M.-R. and Y.-H.S. performed and analysed confocal microscopy experiments in the initial study. A. Galenza and Y.-H.S. performed and analysed confocal microscopy experiments in the revised study. I.K. performed and analysed EM experiments with support from P.M.-R., B.M.H. and C.K., and using guts dissected and fixed by P.M.-R. I.K., P.M.-R., Y.-H.S., A. Galenza and L.A.-A. segmented EM data. A. Guichet and A.D. cloned Meduse and analysed its localization in egg chambers. Y.-H.S. mapped the site of the A142 insertion with technical guidance from J.-M.K. I.K. and B.M.H. supervised EM portions of the project. A. Galenza, J.-M.K. and L.E.O. wrote and revised the manuscript. P.M.-R., Y.-H.S., B.M.H. and I.K. commented on the manuscript. L.E.O. supervised the project.

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Correspondence to Lucy Erin O’Brien.

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Nature Cell Biology thanks Barry Thompson, Bruce Edgar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Same images as Fig. 1b–g, without drawn cell outlines.

a-f, Images are representative of 119 stem cells and 125 enteroblasts across 5 guts. Scale bars, 5 μm, Full genotypes in Supplementary Table 1.

Extended Data Fig. 2 Same images as Fig. 2b,c, showing Su(H)-lacZ expression (b-galactosidase immunostain).

a-b, Multi-channel and β-galactosidase channel view of the same images as Fig. 2b,c. The presence of β-galactosidase in Stage 3 and Stage 4 cells demonstrates that these cells derived recently from enteroblasts. During acquisition of the Stage 3 and 4 images, the gain was increased compared to Stages 1 and 2 to visualize lower levels of β-galactosidase. Arrows in (b) point to a Stage 1 enteroblast next to the Stage 4 pre-enterocyte; at the higher gain necessary to visualize β-galactosidase in the Stage 4 pre-enterocyte, β-galactosidase intensity in the Stage 1 enteroblast is overexposed. Panels (a,b) are representative images collected from 40 guts in 2 independent experiments. Images are projections of short confocal stacks. Scale bars, 5 μm. Full genotypes in Supplementary Table 1.

Extended Data Fig. 3 Criteria for identification of integration stage for differentiating progenitor cells.

Integration stage is assessed by localization of two key markers: (1) apical membrane, and (2) SJ. Cartoons display marker localization for Stages 0–5. Apical membrane, cyan; SJEC-EC, yellow; SJPC-EC, orange; progenitor cell (Su(H)-lacZ+ enteroblast or pre-enterocyte), blue; mature neighbor enterocytes, gray.

Extended Data Fig. 4 The A142 splice trap transposon is inserted into CG2556/meduse, the Drosophila homolog of the mammalian actin bundling protein MISP.

a, Genomic location of the splice trap transposon in the A142 line. The insertion was mapped by inverse PCR and genomic PCR to the large first intron of CG2556, approximately 10.6 kb downstream of the splice site in Exon 1. The transposon is inserted in the proper orientation to capture transcripts from CG2556, which would result in an N-terminal GFP tag on the nearly undisrupted protein (Exon 1 encodes only 7 amino acids, including the initiator Met). CG2556 was previously identified as a homolog of the mammalian Mitotic Interactor and Substrate of PLK1 (aka Mitotic Spindle Positioning, MISP)93. MISP is an actin bundling protein that localizes to the rootlets of mouse and human intestinal microvilli94. The tentacular appearance of the fusion protein in oocytes prompted us to name the gene meduse (mdu). b, Mdu::GFP (cyan) co-localizes with cortical actin filaments (magenta, Rhodamin-phalloidin) in Stage 10 oocytes. Image is representative of 10 oocytes. c, Latrunculin B (LatB) treatment disrupts cortical actin filaments in the oocyte and leads to abrogation of the oocyte Mdu::GFP signal. Note that LatB does not disrupt actin in ring canals; localization of Mdu::GFP to ring canals is visible in Panels (c) and (c′). Image is representative of 10 oocytes. Full genotype in Supplementary Table 1.

Extended Data Fig. 5 PAC integration affects neighboring enterocyte-enterocyte SJ dynamics.

Volumetric images were analyzed from midguts that expressed Su(H)-lacZ and an apical marker (moeABD::GFP or mdu::GFP) and that were immunostained for β-galactosidase and an SJ marker (Ssk or Tsp2a). Full genotypes in Supplementary Table 1. a, Cartoon of the SJ parameters measured at each integration stage for progenitor-associated SJ: λ - SJEC-EC length, ψ - SJPC-EC length, and δ - distance from the basal edge of the SJ to the basal epithelium; and parameters measured for neighbor EC-EC SJ: σ - SJEC-EC length, and δ - distance from the basal edge of the SJ to the basal epithelium. Apical membrane, cyan; progenitor SJEC-EC, yellow; progenitor SJPC-EC, orange; progenitor cell (Su(H)-lacZ+ enteroblast or pre-enterocyte), blue; mature neighbor enterocytes, gray; neighbor SJEC-EC, brown. See Methods for measurement details. In Stage 4 depiction, dashed yellow line represents SJEC-EC that is out-of-plane of the drawing. b-c, Raincloud plots (violin plot on left; boxplot on right) show the indicated measurements for SJs associated with Stage 1–5 progenitor cells (blue; n=30 SJs for each stage) and the SJs associated with neighboring enterocytes (gray; n=60 SJs for each stage, two per each integrating progenitor). (b), Total length of SJ associated with progenitor cell (λ + ψ) compared to length of SJ between neighbor EC-EC (σ). (c), Distance from basal edge of the SJ to the basal epithelium (δ). Boxplots display median as center line, the bounds of the box represent the first and third quartiles, minimum and maximum values shown by whiskers, diamonds indicate outliers. (N=7 guts; n=150 progenitor cells).

Source data

Extended Data Fig. 6 High resolution view of FIB-SEM section shown in Fig. 5d.

30 nm-thick sections were cut with a gallium ion beam at 30 keV and 770 pA. Images were taken with the electron beam at 2 keV, 0.8 nA, 2 μm working distance, 20 μs dwell time, 6144x4096 pixel frame size. Pixel size 9.7 nm. Scale bar, 10 μm. Full genotype in Supplementary Table 1.

Extended Data Fig. 7 EC density is unaffected by SJ knockdown but increases following growth inhibition.

a-d, Knockdown of SJ components in enteroblasts does not affect enterocyte density. (a) Boxplot shows the enterocyte density in R4 region of midguts (N=5 guts per genotype). Boxplots display median as center line, the bounds of the box represent the first and third quartiles, minimum and maximum values shown by whiskers. Each data point represents one midgut. Su(H)ts>GFP versus Su(H)ts>sskRNAi (two-tailed Student’s t-test, p=0.8193), Su(H)ts>GFP versus Su(H)ts>Tsp2aRNAi (two-tailed Student’s t-test, p=0.5718). (b-d), Representative immunofluorescent images of gut epithelia from (a) Su(H)ts>GFP, (b) Su(H)ts>sskRNAi, GFP, and (c) Su(H)ts>Tsp2aRNAi, GFP. Guts immunostained for GFP (blue), the SJ marker Coracle (red), and nuclei (DAPI, grayscale). e-g, Growth inhibition in enteroblasts increases enterocyte density. (e) Boxplot shows the enterocyte density in R4 region of midguts (Su(H)ts>+, N=4 guts; Su(H)ts>tsc1/2, N=2 guts). Boxplots display median as center line, the bounds of the box represent the first and third quartiles, minimum and maximum values shown by whiskers. Each data point represents one midgut. Su(H)ts>+ versus Su(H)ts>tsc1/2 (two-tailed Student’s t-test, p=0.0030). (f,g), Representative immunofluorescent images of gut epithelia from (f) Su(H)ts>+ and (g) Su(H)ts>tsc1/2. Guts immunostained for GFP (blue), the SJ marker Coracle (red), and nuclei (DAPI, grayscale). Scale bars, 25 μm. Full genotypes are in Supplementary Table 1.

Source data

Extended Data Fig. 8 Mechanisms of epithelial cell incorporation.

Three mechanisms to incorporate stem cell progeny into a mature epithelium are shown. Apical membrane (cyan), occluding junction (red), differentiating cell (blue), terminally-differentiated cells (gray), and stem cells (white). a, Symmetric inheritance. Stem cells possess occluding junctions, which are inherited by their progeny. b, Radial intercalation. Stem cells lack occluding junctions. As stem cell progeny differentiate, they grow apically, wedging themselves between terminally-differentiated cells. When they reach the occluding junction of the epithelium, the differentiating cell forms occluding junctions with its neighbors. These junctions expand radially in a ring around the cell’s nascent apical membrane. c, Pre-assembled Apical Compartment (PAC) integration. Stem cells lack occluding junctions Differentiating cells create a transient, occluding junction niche that supports development of the new cell’s future, lumen-facing apical surface. This Pre-assembled Apical Compartment (PAC) is formed from deep, apical plasma membrane pit in the differentiating cell that is covered by overlying mature cells. As the new cell grows and differentiates, the transitional junction mediates a basal-to-apical neighbor exchange between the new cell and mature cells that exposes the PAC to the gut lumen and seamlessly integrates the new cell into the epithelial barrier. d, PACs are asymmetric structures with split apical/basolateral character. The pre-enterocyte’s apical membrane pit accounts for most of the PAC’s surface area.

Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Table 1. List of genotypes of flies used in each figure panel. Supplementary Table 2. Reagents and resources. Supplementary Table 3. Antibody validation.

360° confocal reconstruction of a stage 3 pre-enterocyte shows structure of the PAC (related to Fig. 2b). Video shows reconstructed 360° view of a stage 3 pre-enterocyte, labelled by Su(H)-driven β–galactosidase. The pre-enterocyte is surrounded by two mature enterocytes, and a pair of small, basal progenitor cells is visible between the pre-enterocyte and one of the mature enterocytes. The apical marker MoeABD::GFP outlines the lumenal-apical surface of the mature enterocytes, the PAC in the pre-enterocyte, and the entire cortex of the progenitor cells. The SJ protein Tetraspanin2A (Tsp2a) forms a convex shroud that covers the apex of the pre-enterocyte. Nuclei are labelled with DAPI. Full genotype in Supplementary Table 1.

CLEM, FIB-SEM dataset and animated volumetric rendering of stage 1 enteroblast, neighbour enterocytes and nascent transitional SJ (related to Fig. 5b,c). CLEM image and tomographic reconstruction of Stage 1 enteroblast from a Su(H)-GFP:nls midgut. Volume reconstructed from 30 serial sections. Sections were cut with a gallium ion beam at 10 kV, spot size 5, pixel frame size 4,096 × 4,096, pixel dwell time 10 μs, pixel size 8.7 nm, slice thickness, 150 nm, volume of reconstruction 35.6 μm × 35.6 μm × 4.5 μm. Full genotype in Supplementary Table 1.

FIB-SEM dataset and animated volumetric rendering of stage 2 enteroblast, neighbour cells and transitional SJ (related to Fig. 5d,e). Tomographic reconstruction of stage 2 enteroblast from 415 serial ultrathin FIB-SEM sections, including the image shown in Fig. 5d and Extended Data Fig. 6. Volume of reconstruction, 55 μm × 36.6 μm × 12.3 μm. Slice thickness, 30 nm. Full genotype in Supplementary Table 1.

3D ultrastructure of PAC, PAC precursor and their associated pre-enterocyte (related to Fig. 7a–d). Tomographic reconstruction of 200 serial FIB-SEM images, including a cropped version of the image shown in Fig. 7a. Rotation of 360° reveals the ellipsoid and allantoid shapes of the PAC and PAC precursor, respectively, and also reveals holes in the SJ in which the pre-enterocyte and enterocyte membranes have separated to form the intercellular lumens. Volume of reconstruction: 40.2 μm × 23.9 μm × 8 μm. Slice thickness, 40 nm. Full genotype in Supplementary Table 1.

Source data

Source Data Fig. 1

Source data for Fig. 1h.

Source Data Fig. 2

Source data for Fig. 2d.

Source Data Fig. 3

Source data for Fig. 3d.

Source Data Fig. 4

Source data for Fig. 4.

Source Data Fig. 6

Source data for Fig. 6a,f.

Source Data Extended Data Fig. 5

Source data for Extended Data Fig. 5.

Source Data Extended Data Fig. 7

Source data for Extended Data Fig. 7.

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Galenza, A., Moreno-Roman, P., Su, YH. et al. Basal stem cell progeny establish their apical surface in a junctional niche during turnover of an adult barrier epithelium. Nat Cell Biol 25, 658–671 (2023). https://doi.org/10.1038/s41556-023-01116-w

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