De novo apical domain formation inside the Drosophila adult midgut epithelium
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Evaluation Summary:
This paper addresses a fundamental cell biological question - the de-novo development of an apical membrane during the integration of an initially unpolarized cell, the enterocyst, into an an existing epithelium, the Drosophila midgut. The data will be of interest to a wide range of researchers including those in the fields of cell, development, stem cell and cancer biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
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.
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Author Response
Reviewer #1 (Public Review):
This paper is a follow-up of the authors previous paper (2018), in which they carefully described the organisation of the junctions between cells of the adult Drosophila midgut epithelium and their control from the basal side by integrin signalling. Here, the authors used state-of-the art imaging and genetics to unravel step-by-step the events leading from an initially unpolarised cell to an epithelial cell that integrates into the existing epithelium. Many of the images are accompanied by cartoons, which help the reader to better understand the images and follow the conclusions. It would have been helpful yet, in particular with respect to the mutant phenotypes described later, if they would have named each of the steps/stages. In addition, mentioning the timescale would give an idea …
Author Response
Reviewer #1 (Public Review):
This paper is a follow-up of the authors previous paper (2018), in which they carefully described the organisation of the junctions between cells of the adult Drosophila midgut epithelium and their control from the basal side by integrin signalling. Here, the authors used state-of-the art imaging and genetics to unravel step-by-step the events leading from an initially unpolarised cell to an epithelial cell that integrates into the existing epithelium. Many of the images are accompanied by cartoons, which help the reader to better understand the images and follow the conclusions. It would have been helpful yet, in particular with respect to the mutant phenotypes described later, if they would have named each of the steps/stages. In addition, mentioning the timescale would give an idea about the temporal frame in which this process elapses.
We have used terms such as “unpolarised cells, polarised Actin/Cno” to label different stages in Figure 6, since this sequence of steps is inferred from results obtained from fixed samples with still images. We have illustrated the septate junction mutant phenotype in Figure 8I.
We have also performed a new experiment to estimate the time taken for an activated EB to form a PAC and to become a mature enterocyte using overexpressing Sox21a with esg[ts]>GFP to induce enteroblast differentiation. Counting the number of GFP+ve cells without PAC, with a PAC and with full apical domain at different time points suggests that activated EBs take about a day to form a PAC and another day to form a fully-integrated enterocyte. We have summarised the results in Figure 5-figure supplement 1C.
We have also included this result in the main-text as “ To estimate the time taken for enteroblasts to progress to pre-enterocytes with a PAC, and for pre-enterocytes become to enterocytes, we induced enterocyte differentiation by over-expressing UAS-Sox21a under the control of esg[ts]-Gal4 and counted the number of GFP+ve cells without a PAC or apical domain, with a PAC and with a full apical domain at different time points after induction (Chen et al., 2016; Meng and Biteau, 2015; Zhai et al., 2017). 17 hours after shifting the flies to 25ºC to inactivate Gal80ts, almost no GFP+ve cells had progressed to pre-EC with a PAC (0.1%) or EC (1%), and these few cells probably started to differentiate before Sox 21a induction. 24 hours later, 10% of the GFP+ve cells had developed into pre-ECs with a PAC and 20% had become ECs (Figure 5-figure supplement 1B-C). After an additional 24 hours, the number of cells with a PAC fell to 1%, whereas 50% were ECs. Assuming that it takes 12-17 hours to induce high levels of Sox21a expression, these results suggest that most activated EBs take about 24 hours to develop into a pre-EC with a PAC and a further 24 hours to differentiate into a mature EC, although some cells differentiate faster. This time frame is in agreement with a previous study using similar approaches to accelerate differentiation (Rojas Villa et al., 2019) and a recent live imaging study tracing the enteroblast to enterocyte transition (Tang et al., 2021). These results also indicate that down-regulation of Sox21a is not essential for enteroblast to pre-enterocyte differentiation, since enteroblasts overexpressing Sox21a still from a PAC (Figure 5-figure supplement 1B).
The authors convincingly show that septate junctions are instrumental for proper polarisation and integration of the enteroblast. However, while they nicely showed that Canoe in neither required in the enteroblast nor in the enterocytes for this process, it remains unclear whether septate junction proteins are required in enteroblast or in enterocytes or in both and at which particular step the process fails in the mutant.
Early stage enteroblasts neither express or require septate junction proteins, whereas late stage enteroblasts and pre-enterocytes do (Chen et al., 2020; Hung et al., 2020; Izumi et al., 2019; Xu et al., 2019). Since cells mutant for septate junction proteins do not develop into mature enterocytes with an apical domain facing the gut lumen, we cannot answer the reviewer’s question of whether septate junction proteins are required in enterocytes.
As we discussed in the paper, we think that “differentiating enteroblasts only require a basal cue to establish their initial apical-basal polarity, whereas the formation of the pre-assembled apical compartment also requires a junctional cue. The septate junctions are not necessary for apical domain formation per se, however, as mesh mutant enteroblasts form a full-developed apical domain with a brush border inside the cell. This suggests that septate junctions define the site of apical domain formation by delimiting the region where apical membrane proteins are secreted to assemble the brush border, but do not control the process of apical domain formation directly.”
Reviewer #2 (Public Review):
The authors recently showed the polarization of the cells of the adult Drosophila midgut does not require any of the canonical epithelial polarity factors, and instead depend on basal cues from adhesion to the ECM, as well as septate junction proteins (Chen et al, 2018). Here they extend this research to examine in greater detail precisely how midgut epithelial cells integrate in the pre-exisiting epithelium and become polarized. Surprisingly, they show that enteroblasts form an apical membrane initiation site prior to polarizing. Furthermore, they show that this develops into a pre-apical compartment containing fully-formed brush border. This is a very interesting finding - it explains how integrating enteroblasts can integrate into a pre-existing epithelium without disrupting barrier function. The conclusions of this paper are mostly well supported by data, but some aspects could do with being clarified and extended as outlined below.
Model presented in Figure 6
While the separation of membranes indicated in Figure 6 steps 3-5 can be seen in the image shown in Figure 3B, this is one of the only images which supports the idea that there is a separation of membranes between the enteroblast and overlying enterocytes during PAC formation. Is the model in Figure 6 supported by EM data - can you see a region where there is brush border and separation of cells? Supplementing Figure 3 with corresponding EM images would greatly aid the reader in interpreting the data and strengthen the model.
We think that AJ clearing and membrane separation is a brief process that is quickly followed by the separation of the apical and junctional proteins and apical secretion at the AMIS to form the PAC. We have not captured this stage in our EM images, but have many other examples that show this step (e.g Figure 4C and Figure 8F). Another example is shown below.
A key step in the model is that the clearance of E-Cadherin from the apical membrane leads to a loss of adhesion between the enteroblast and the overlying enterocytes. This would need to be supported by functional data such as overexpression of E-Cad or E-CadDN in enteroblasts or by generating shg mutant clones. If the model is correct, perturbing E-Cad levels in enteroblasts should lead to defects in PAC formation, such as loss of de-adhesion/early de-adhesion/excessive de-adhesion.
We think it is the local clearance of ECad from the apical membrane, not the downregulation of total level of ECad that is important for the local membrane separation and future PAC formation. The experiment of overexpressing ECad or ECad-DN proposed by the reviewer might be crucial to demonstrate the importance of total amount of ECad, but might not be very helpful in determining the importance of membrane separation in the PAC formation. Moreover, AJ formation in fly midgut epithelium does not depend on ECad, suggesting that ECad and NCad act redundantly which further complicates this approach (Choi et al., 2011; Liang et al., 2017).
Role for the septate junction proteins
Septate junction proteins were previously shown by these authors to be required for enteroblast polarization and integration into the midgut epithelium (Chen et al, 2018). Here they extend this by examining enteroblasts mutant for septate junction proteins, and conclude that septate junction proteins are required for normal PAC formation. However, it is not clear what aspect of the polarization of the enteroblasts is disrupted, because a number of mesh mutant cells (albeit a lower proportion than in wildtype) do form PACs. The main phenotype seems to be that cells fail to polarize (as previously reported) or have internalised PACs. It is hard to know what to conclude from this data about the role of the septate junction components in PAC formation.
The major phenotype of the septate junction mutants is the loss of polarity, i.e. an inability to form an apical domain and integrate into the epithelial layer as shown in Figure 8. Neither mesh or Tsp2a mutants can form a PAC, even though mesh mutant cells have higher propensity to form an internal PAC-like structure (Figure 8B,C,E,G,H, Figure 8-figure supplement 1L). Thus, we think that septate junctions are required for AMIS and PAC formation. What complicates the interpretation is that some (6-20%) septate junction mutant cells do form an AMIS like structure (Figure 8D-F, Figure 8-figure supplement 1F&K). The simplest explanation for this result is that this is due to perdurance of the wild-type proteins after clone induction, with the weaker phenotype of ssk mutants being due to longer perdurance of this protein. However, we cannot rule out the alternative explanation that AMIS and PAC formation is facilitated by the septate junction proteins, but that they can still form very inefficiently in their absence.
We realise that this section was quite confusing in the orginal version of the manuscript and have now re-written it to make this interpretation clearer.
Coracle is used as a readout for the localization of septate junction components, yet the staining for Cora in Figure S3B looks quite different to Mesh in S3D. If Cora is to be used as a readout for the localization of septate junction components, then staining for Cora/Mesh and/or Cora/SSk or Tsp2a should be shown.
When discussing the requirement for septate junctions for enteroblast integration - Coracle and Mesh are used interchangeably - but as mentioned before, it is not clear if they colocalize, or if their localization is interdependent (as demonstrated for Mesh, Tsp2a and Ssk in Figure 7). What is the phenotype of enteroblasts mutant for cora?
Following from the previous point - while it is clear that Coracle is apical early during AMIS formation, it is not clear if Mesh, Tsp2a and Ssk also are, yet these are the mutants that are examined for a role in AMIS/PAC formation. It would be good to know whether the loss of cora would lead to defects in AMIS formation.
The reason we used mainly Coracle as a marker for the septate junctions is that Mesh and Tsp2A localise to the basal labyrinth as well as to the septate junctions which could confuse the reader. We have now added new panels to Figure 3-figure supplement 3E&F showing the colocalization of Cora with Mesh/Tsp2a at the septate junctions and during the crucial stages of PAC formation.
Additional Results:
"Coracle is a peripheral septate junction protein whose localisation depends on the structural septate junction components such as Mesh/Ssk/Tsp2a (Chen et al., 2018; Izumi et al., 2016, 2012). Cora antibody staining provides a clearer marker for the septate junctions than Mesh or Tsp2a antibody staining, because the latter also label the basal labyrinth (Figure 3-figure supplement 1E&F). To determine whether Cora is required for PAC formation or epithelial polarity in the adult midgut, we generated a null mutant allele with a premature stop codon in FERM domain using CRISPR. Cells mutant for this allele, corajc, or a second cora null allele, cora5, can form a PAC, septate junctions and a full apical domain, indicating that Cora is also not required for enteroblast integration or enterocyte polarity (Figure 7F&G, Figure 7-figure supplement 1E-H).
Additional Materials and Methods:
We used the CRISPR/Cas9 method (Bassett and Liu, 2014) to generate null alleles of canoe and coracle. sgRNA was in vitro transcribed from a DNA template created by PCR from two partially complementary primers:
forward primer:
For coracle:
5′-GAAATTAATACGACTCACTATAGAAGCTGGCCATGTACGGCGGTTTTAGAGCTAGAAATAGC-3′;
The sgRNA was injected into…Act5c-Cas9 embryos to generate coracle null alleles (Port et al., 2014). Putative…coracle mutants in the progeny of the injected embryos were recovered, balanced, and sequenced. …The coraclejc allele contains a 2bp deletion around the CRISPR site, resulting in a frameshift that leads to stop codon at amino acid 225 in the middle of the FERM domain, which is shared by all isoforms. No Coracle protein was detectable by antibody (DSHB C615.16) staining in both midgut and follicle cell clones. The coraclejc allele was recombined with FRT G13 to make the FRTG13 coraclejc flies.
It is unclear what is happening in Figure 8A,C,E, S7D. Is that a detachment phenotype or an integration phenotype? Are the majority of cells unpolarised due to loss of integrin attachment rather than failure to form an AMIS/PAC?
Cells mutant for septate junction proteins do not detach from the basement membrane and still localise Talin basally, as illustrated by the new panel we have added (Figure 8-figure supplement 1N), showing Talin localisation in Tsp2a mutant cell.
However, because the mutant cells cannot integrate and remain stuck beneath the septate junctions between the enterocytes, they sometimes become displaced from a portion of the basement membrane by younger EBs that derive from the same mutant ISC, leading to a pile up of cells in the basal region of the epithelium (e.g. Figure 8A, E and H).
We have added the following sentences to the Results, explaining these points:
"Because the mutant cells remain trapped beneath enterocyte-enterocyte septate junctions, they accumulate in the basal region of the epithelium, with new EBs derived from the same mutant ISC forming beneath them and reducing their contact with the basement membrane (Figure 8A)."
" The majority of cells mutant for septate junction components fail to polarise or form an AMIS, although they form normal lateral and basal domains, as the basal integrin signalling component, Talin, localises normally (Figure 8-figure supplement 1N)."
It is unclear whether enteroblasts really pass through an 'unpolarized stage'. In Figure 6, when they are described as 'unpolarised', they clearly have distinct basal and AJ domains. In septate junction mutants, when cells are classified as unpolarized, do they still have distinct regions of integrin/E-Cad expression?
This is a semantic question. We agree that they have distinct lateral and basal domains, but they do not have an apical domain. In this respect, these "unpolarised" cells are similar to a mesenchymal fibroblast migrating on a substrate, which has a distinct basal side contacting the substrate that is different from the non-contacting regions of the cell surface. They also match the description of the migratory, "mesenchymal" enteroblasts (Antonello et al., 2015). To make this clearer, we have added the following notes to the legend for Figure 6: “Unpolarised” in the second panel of this figure indicates that the enteroblast has not formed a distinct apical domain. At this stage, no marker is clearly apically localised. “unpolarised” or “polarised” in the third and fourth panels describe the localisation of marker proteins, such as Actin and Cno."
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Evaluation Summary:
This paper addresses a fundamental cell biological question - the de-novo development of an apical membrane during the integration of an initially unpolarized cell, the enterocyst, into an an existing epithelium, the Drosophila midgut. The data will be of interest to a wide range of researchers including those in the fields of cell, development, stem cell and cancer biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
1. This manuscript presents data to show how an initially unpolarised cell, the intestinal stem cell/enteroblast in the adult Drosophila midgut, acquires an apical pole de-novo during its integration into the existing epithelium. Results show the formation of an actin-rich "Preformed Apical Compartment" (PAC) on the enteroblast, which ultimately leads to the formation of an apical membrane during differentiation of the enteroblast to an enterocyte. During the integration into the epithelium, existing septate junctions between two neighbouring enterocytes are replaced by newly formed septate junctions between the differentiating enteroblast and the enterocyte, thus maintaining a functional barrier throughout this process. Data presented provide further evidence that septate junctions are instrumental to …
Reviewer #1 (Public Review):
1. This manuscript presents data to show how an initially unpolarised cell, the intestinal stem cell/enteroblast in the adult Drosophila midgut, acquires an apical pole de-novo during its integration into the existing epithelium. Results show the formation of an actin-rich "Preformed Apical Compartment" (PAC) on the enteroblast, which ultimately leads to the formation of an apical membrane during differentiation of the enteroblast to an enterocyte. During the integration into the epithelium, existing septate junctions between two neighbouring enterocytes are replaced by newly formed septate junctions between the differentiating enteroblast and the enterocyte, thus maintaining a functional barrier throughout this process. Data presented provide further evidence that septate junctions are instrumental to initiate the apical membrane in enteroblasts and ensure their proper integration.
2. This paper is a follow-up of the authors previous paper (2018), in which they carefully described the organisation of the junctions between cells of the adult Drosophila midgut epithelium and their control from the basal side by integrin signalling. Here, the authors used state-of-the art imaging and genetics to unravel step-by-step the events leading from an initially unpolarised cell to an epithelial cell that integrates into the existing epithelium. Many of the images are accompanied by cartoons, which help the reader to better understand the images and follow the conclusions. It would have been helpful yet, in particular with respect to the mutant phenotypes described later, if they would have named each of the steps/stages. In addition, mentioning the timescale would give an idea about the temporal frame in which this process elapses.
The authors convincingly show that septate junctions are instrumental for proper polarisation and integration of the enteroblast. However, while they nicely showed that Canoe in neither required in the enteroblast nor in the enterocytes for this process, it remains unclear whether septate junction proteins are required in enteroblast or in enterocytes or in both and at which particular step the process fails in the mutant.
3. Most of the data allow the reader to follow the conclusions made. The data presented extend our current knowledge by showing a novel mechanism by which a cell can acquire an apical pole de-novo. The adult fly midgut is an ideal system to analyse this process is an organ (rather than in cells in culture).
4. Given the high degree of similarity between the adult Drosophila midgut and the mammalian small intestine, the data presented here will certainly stimulate novel concepts and approaches to unravel mechanisms involved in the maintenance of epithelial homeostasis, not only in flies, but also in mammals. Loss of homeostasis can lead to various diseases, including cancer.
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Reviewer #2 (Public Review):
The authors recently showed the polarization of the cells of the adult Drosophila midgut does not require any of the canonical epithelial polarity factors, and instead depend on basal cues from adhesion to the ECM, as well as septate junction proteins (Chen et al, 2018). Here they extend this research to examine in greater detail precisely how midgut epithelial cells integrate in the pre-exisiting epithelium and become polarized. Surprisingly, they show that enteroblasts form an apical membrane initiation site prior to polarizing. Furthermore, they show that this develops into a pre-apical compartment containing fully-formed brush border. This is a very interesting finding - it explains how integrating enteroblasts can integrate into a pre-existing epithelium without disrupting barrier function. The …
Reviewer #2 (Public Review):
The authors recently showed the polarization of the cells of the adult Drosophila midgut does not require any of the canonical epithelial polarity factors, and instead depend on basal cues from adhesion to the ECM, as well as septate junction proteins (Chen et al, 2018). Here they extend this research to examine in greater detail precisely how midgut epithelial cells integrate in the pre-exisiting epithelium and become polarized. Surprisingly, they show that enteroblasts form an apical membrane initiation site prior to polarizing. Furthermore, they show that this develops into a pre-apical compartment containing fully-formed brush border. This is a very interesting finding - it explains how integrating enteroblasts can integrate into a pre-existing epithelium without disrupting barrier function. The conclusions of this paper are mostly well supported by data, but some aspects could do with being clarified and extended as outlined below.
Model presented in Figure 6:
While the separation of membranes indicated in Figure 6 steps 3-5 can be seen in the image shown in Figure 3B, this is one of the only images which supports the idea that there is a separation of membranes between the enteroblast and overlying enterocytes during PAC formation. Is the model in Figure 6 supported by EM data - can you see a region where there is brush border and separation of cells? Supplementing Figure 3 with corresponding EM images would greatly aid the reader in interpreting the data and strengthen the model.
Coracle is used as a readout for the localization of septate junction components, yet the staining for Cora in Figure S3B looks quite different to Mesh in S3D. If Cora is to be used as a readout for the localization of septate junction components, then staining for Cora/Mesh and/or Cora/SSk or Tsp2a should be shown.
A key step in the model is that the clearance of E-Cadherin from the apical membrane leads to a loss of adhesion between the enteroblast and the overlying enterocytes. This would need to be supported by functional data such as overexpression of E-Cad or E-CadDN in enteroblasts or by generating shg mutant clones. If the model is correct, perturbing E-Cad levels in enteroblasts should lead to defects in PAC formation, such as loss of de-adhesion/early de-adhesion/excessive de-adhesion.
Role for the septate junction proteins:
Septate junction proteins were previously shown by these authors to be required for enteroblast polarization and integration into the midgut epithelium (Chen et al, 2018). Here they extend this by examining enteroblasts mutant for septate junction proteins, and conclude that septate junction proteins are required for normal PAC formation. However, it is not clear what aspect of the polarization of the enteroblasts is disrupted, because a number of mesh mutant cells (albeit a lower proportion than in wildtype) do form PACs. The main phenotype seems to be that cells fail to polarize (as previously reported) or have internalised PACs. It is hard to know what to conclude from this data about the role of the septate junction components in PAC formation.
When discussing the requirement for septate junctions for enteroblast integration - Coracle and Mesh are used interchangeably - but as mentioned before, it is not clear if they colocalize, or if their localization is interdependent (as demonstrated for Mesh, Tsp2a and Ssk in Figure 7). What is the phenotype of enteroblasts mutant for cora?
Following from the previous point - while it is clear that Coracle is apical early during AMIS formation, it is not clear if Mesh, Tsp2a and Ssk also are, yet these are the mutants that are examined for a role in AMIS/PAC formation. It would be good to know whether the loss of cora would lead to defects in AMIS formation.
It is unclear what is happening in Figure 8A,C,E, S7D. Is that a detachment phenotype or an integration phenotype? Are the majority of cells unpolarised due to loss of integrin attachment rather than failure to form an AMIS/PAC?
It is unclear whether enteroblasts really pass through an 'unpolarized stage'. In Figure 6, when they are described as 'unpolarised', they clearly have distinct basal and AJ domains. In septate junction mutants, when cells are classified as unpolarized, do they still have distinct regions of integrin/E-Cad expression?
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Reviewer #3 (Public Review):
In this interesting paper, Chen & St. Johnston deliver a detailed description of how newborn enterocytes (ECs) integrate into the gut epithelium of Drosophila. They show that this involves a novel mechanism whereby a new apical plasma membrane domain is formed inside the cell, before it is exposed to the gut lumen, and they provide extensive data on the localizations and movements of many cell adhesion and cytoskeletal proteins involved in the process. Their description of this unusual process is complete, clear, and convincing, even though their analysis used exclusively fixed samples and lacked a real-time analysis (which would be technically very challenging, if not impossible at this time). The description of EC integration is significant not only for the unique cell biology at play, but because it …
Reviewer #3 (Public Review):
In this interesting paper, Chen & St. Johnston deliver a detailed description of how newborn enterocytes (ECs) integrate into the gut epithelium of Drosophila. They show that this involves a novel mechanism whereby a new apical plasma membrane domain is formed inside the cell, before it is exposed to the gut lumen, and they provide extensive data on the localizations and movements of many cell adhesion and cytoskeletal proteins involved in the process. Their description of this unusual process is complete, clear, and convincing, even though their analysis used exclusively fixed samples and lacked a real-time analysis (which would be technically very challenging, if not impossible at this time). The description of EC integration is significant not only for the unique cell biology at play, but because it explains how new cells are integrated into the gut epithelium without breaching the gut's barrier function. Aspects of this process may well prove relevant to cell integration in other endodermal barrier epithelia that are renewed by stem cells. Overall the paper is a very high quality work that should be valuable in the field of Drosophila gut homeostasis, and more broadly as a fine example of epithelial cell biology. Moreover, the manuscript is very well written, easy to follow, and the illustrations and data are all very good.
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