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. 2026 Aug 3;118(8):e70076. doi: 10.1111/boc.70076

AP‐1B Controls Several Levels of Cell Proliferation and Differentiation in Mouse Enteroids

Maela Duclos 1, Anne Bourdais 1, Ophélie Nicolle 1, Alexandre Helpiquet 1, Grégoire Michaux 1,✉, Aurélien Bidaud‐Meynard 1,✉
PMCID: PMC13430592  PMID: 42544834

ABSTRACT

Gut homeostasis relies on the tightly controlled balance between intestinal stem cell self‐renewal and differentiation. The clathrin adaptor complex AP‐1B plays a pivotal role in establishing the polarity of enterocytes as well as in the asymmetric distribution of membrane proteins, including the brush border transporters that govern intestinal absorption. Additionally, AP‐1B has been involved in the control of intestinal cell proliferation, which suggests that it may regulate various aspects of gut functional organisation. In this study, we investigated the consequences of conditional mutations of the gene encoding the AP‐1B subunit μ1B (Ap1m2) in mouse enteroids. We first showed in this model that Ap1m2 mutations also induce strong polarity defects at the subcellular level in the absorptive enterocytes. Next, we unveiled that AP‐1B regulates intestinal cell differentiation at the tissular level, through the commitment of stem/progenitor cells towards the secretory lineage and cell positioning along the crypt‐villus axis. Furthermore, we showed that AP‐1B inhibition also induces hyperproliferation in enteroids. Notably, we unravelled that, complementary to the Wnt/β‐catenin‐mediated proliferation described in null mice, AP‐1B downregulation triggers a tissue‐autonomous, mTOR/YAP‐dependent, proliferative pathway. Overall, these results enlighten the pleiotropic roles played by AP‐1B in the homeostasis of the gut epithelium.

Keywords: AP‐1B, differentiation, epithelia, intestine, polarised trafficking

We show that in mouse intestinal organoids, AP‐1B is implicated in apical polarity, differentiation in the secretory lineage and proliferation. In particular, we propose that proliferation is controlled by AP‐1B via the mTOR and YAP pathways. Arrows show the functional links that were elucidated in this study, and dotted lines show putative downstream functional interactors based on the literature.

graphic file with name BOC-118-e70076-g001.jpg

Abbreviations

AP‐1

Adaptor protein complex 1

APC

Adenomatous polyposis coli

ARH

Autosomal recessive hypercholesterolemia protein

CTGF

Connective tissue growth factor

EGF

Epidermal growth factor

ISC

Intestinal stem cells

mTOR

Mechanistic target of rapamycin

PMP22

Peripheral myelin protein 22

TGN

Trans‐Golgi network

YAP

Yes‐associated protein 1

1. Introduction

Intestinal epithelial cells are constantly replaced by the treadmilling of cells arising from the self‐renewal of Lgr5+ intestinal stem cells (ISCs, aka crypt‐based columnar cells) and anoikis of terminally‐differentiated cells at the tip of the villus. In the crypt, ISC proliferation is controlled by niche factors emanating from neighbouring Paneth cells and stromal cells, such as Wnt pathway agonists and epidermal growth factor (EGF) (Gehart and Clevers 2019). Daughter cells migrating through the transit‐amplifying zone towards the villus are exposed to various environmental gradients (e.g., Wnt, Notch and BMP), which result in their differentiation into secretory (i.e., Paneth, goblet or enteroendocrine) and absorptive lineages (Beumer and Clevers 2021). The most prevalent absorptive cell type, the enterocyte, exhibits an apical array of densely packed microvilli—the so‐called brush border—which increases the surface area available for nutrient absorption and provides a barrier against pathogenic microorganisms (Delacour et al. 2016).

Membrane trafficking is instrumental in key steps of epithelial tissue establishment, such as stem cell asymmetric division, fate acquisition, and differentiation, notably by regulating morphogene‐dependent pathways (Li et al. 2023). In the gut, several trafficking factors have been implicated in both ISC division (e.g., Rab11, Myo5b, Sara endosomes or SH3PX1) and differentiation (e.g., endotubin, Myo5b or Beclin1) (Cox et al. 2018; Goswami et al. 2021; Kaji et al. 2021; Montagne and Gonzalez‐Gaitan 2014; Tran et al. 2024; Weis et al. 2015; Zhang et al. 2019). Furthermore, polarised trafficking is also involved in the functional organisation of the epithelia by regulating apical–basal polarity, the onset of the apical membrane initiation site and luminogenesis, as well as brush border maintenance (Apodaca et al. 2012; Bidaud‐Meynard et al. 2019; Bryant et al. 2010; Knowles et al. 2014).

Adaptor protein complex 1 (AP‐1) is a tetrameric complex made of two large subunits (β1 and γ), one medium subunit (μ1A or μ1B) and one small subunit (σ1) (Nakatsu et al. 2014). This complex, through its clathrin adaptor function, handles the polarised sorting of cargoes from the trans‐Golgi network (TGN) and recycling endosomes to the plasma membrane (Guo et al. 2013), as well as the retrograde trafficking to the TGN (Robinson et al. 2024). Although the specific localisation and the degree of complementarity/redundancy between AP‐1A (made of μ1A subunit) and AP‐1B (made of μ1B subunit) complexes are still unclear (Fölsch et al. 2003; Rodriguez‐Boulan et al. 2013; Ruturaj et al. 2023), the function of the epithelium‐specific AP‐1B complex in cell polarity has been clearly established (Fölsch 2015). Indeed, extensive studies have shown that AP‐1B is responsible for the basolateral sorting of cargoes bearing di‐leucine or tyrosine‐based signals (Fölsch et al. 1999; Fölsch et al. 2001; Gan et al. 2002; Gravotta et al. 2007), as well as FxNPxY signals, in cooperation with the autosomal recessive hypercholesterolemia protein (ARH) (Kang and Fölsch 2011). Furthermore, it has been demonstrated that the AP‐1 complex also mediates apical sorting in Caenorhabditis elegans and mouse intestine models, as well as in cultured cells (Caceres et al. 2019; Hase et al. 2013; Shafaq‐Zadah et al. 2012; Zhang et al. 2012). In enterocytes, besides the contralateral localisation of integral proteins, AP‐1 deficiency resulted in the basolateral accumulation of apical polarity determinants, leading to the apicalisation of the lateral membrane (i.e., ectopic lumen) (Hase et al. 2013; Shafaq‐Zadah et al. 2012), as well as to intestinal barrier failure (Klee et al. 2020). Another polarised process in the gut, immune response, has also been shown to rely on AP‐1B function (Matsumoto et al. 2024; Stanifer et al. 2020). Finally, mutations of AP‐1 subunits have been linked to several enteropathies, such as MEDNIK (Mental disability, Enteropathy, Deafness, Neuropathy, Ichthyosis and Keratodermia) and KIDAR (Keratitis, Icthyosis, Deafness and Autosomal Recessive) diseases, which underscore its significance in intestinal homeostasis (Szabó et al. 2024).

Noticeably, it has been shown that organismal knockout (KO) of the gene coding the AP‐1B subunit μ1B (Ap1m2) in mouse provokes crypt hyperplasia, while μ1B expression is downregulated in an adenomatous polyposis coli (APC)‐deficient intestinal tumour model (Hase et al. 2013; Mimura et al. 2012). These observations suggested that, in addition to cell polarity, AP‐1B complex controls intestinal cell proliferation. In this study, we mutated the Ap1m2 gene in mouse enteroids to specifically assess the extent of the roles played by AP‐1B in the intestinal epithelium.

2. Results and Discussion

2.1. Ap1m2 Inactivating Mutations Cause Strong Polarity Defects in Enterocytes

The Ap1m2 gene was mutated by conditional CRISPR‐Cas9 in adult stem cell‐derived mouse intestinal organoids (aka enteroids) as described before (Bidaud‐Meynard et al. 2024; Saleh et al. 2023). Sanger sequencing of the genomic DNA of edited enteroids using Inference CRISPR Edits (ICE) software (Conant et al. 2022) showed that 80% of Ap1m2 crispants were mutated (hereafter called Ap1m2mut ), with half of the mutations consisting of a single deletion of a guanosine located 5′ of the protospacer adjacent motif (PAM), which leads to a frameshift at amino acid 105 and a premature stop codon at amino acid 200 (out of 425) (Figures 1A and S1A,B). Because of the absence of suitable antibodies, the effect of Ap1m2 mutations on AP‐1B protein expression could not be directly tested. However, we observed that these mutations resulted in a 38 ± 21% decrease of Ap1m2 transcript, as measured by RT‐qPCR (Figure S1C). To first validate our knockdown strategy and confirm the sorting function of AP‐1B in fully polarised intestinal cells, villus‐like terminal differentiation of the enterocytes was induced by supplementation of the medium with valproic acid and inhibitor of Wnt production 2 (IWP2) (Mosa et al. 2018). Ap1m2mut enteroids indeed displayed a partial epithelial mispolarisation, as evidenced by the lateral accumulation of the brush border membrane‐F‐actin crosslinker phospho‐ezrin (Figure 1B,C). This was substantiated by analysing Ap1m2mut enteroids by transmission electron microscopy (TEM), which exhibited a normal apical brush border but often displayed microvilli at the lateral or basal poles (Figure 1D‐F), thus fully recapitulating the results obtained in C. elegans and mouse (Hase et al. 2013; Shafaq‐Zadah et al. 2012). Interestingly, while disturbing polarized trafficking by overexpressing EMK1, the ortholog of the C. elegans polarity determinant PAR‐1, induced a full switch of cell polarity with a relocalisation of the lumen to the lateral side (i.e., columnar‐to‐hepatic phenotype) (Cohen et al. 2004a; Cohen et al. 2004b), Ap1m2 inactivation rather induced a loss of polarity with a brush border at both the apical and basolateral pole. The similarity of this very specific polarity phenotype relocalising apical structures to the lateral membrane observed in vivo in two evolutionarily distant models validated our AP‐1B inactivation strategy. We next studied these polarity defects in proliferating enteroids maintained in classical EGF/Noggin/R‐Spondin1 (ENR) medium (Sato et al. 2011). Proliferating enteroids also presented ectopic lateral lumens, and 21 % of them displayed an apical mislocalisation of the basolateral protein EphB2 (Figure 1G,H). Our previous work in C. elegans uncovered that these polarity defects relied on the cytoplasmic dispersion of Rab11+ recycling endosomes and the subsequent mistargeting of the apical polarity determinant Cdc42, thus recruiting the other members of the PAR module at the lateral membrane to convert it into an apical pole (Shafaq‐Zadah et al. 2012). As already shown in mouse as well as in human and C. elegans intestine (Bidaud‐Meynard et al. 2019; Dooley et al. 2025; Vogel et al. 2017), the vast majority of Rab11+ endosomes accumulate in the vicinity of the apex and can be marginally found as dot‐like structures (Figures 1I and S1D). Quantification of apical Rab11 signal showed a decreased apical/cytoplasmic ratio in Ap1m2mut enteroids compared to controls (1.18 ± 0.15 vs. 1.46 ± 0.27, Figure 1J), supporting the idea that AP‐1B controls a similar pathway in C. elegans and mammals. Moreover, TEM of Ap1m2mut enteroids revealed dilated Golgi cisternae and abundant associated vesicles (Figure S1E). Overall, these strong subcellular defects substantiate the key role of AP‐1B‐mediated trafficking in the apical–basal polarity of enterocytes and the dual role of this complex in both apical and basolateral sorting.

FIGURE 1.

FIGURE 1

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Ap1m2 mutations in enteroids lead to strong polarity defects in enterocytes. (A) The histogram shows the repartition of indels found in the enteroid population (n = 3 independent experiments). (B,C) Phospho‐ezrin (P‐ezrin) staining in fully‐differentiated control and Ap1m2mut enteroids. Arrowheads indicate the lateral localisation of P‐ezrin. (C) Quantification of the lateral+cytoplasmic/cytoplasmic ratio of P‐ezrin signal (n = 6–12 enteroids per condition per experiment (coloured dots) from 4 independent experiments, Mann–Whitney test). (D–G) TEM analysis of fully‐differentiated (D–F) or proliferating (G) control and Ap1m2mut enteroids. The right panel in F is a higher magnification of the ROI, showing basal microvilli. In (E) and (G), the ectopic lumens are connected to the lateral membrane by junctions, pointed by arrowheads. (H) Immunostaining of EphB2 in control and Ap1m2mut enteroid crypts. The arrow shows EphB2 at the apical pole, n = 6–12 enteroids analysed per experiment, from 4 independent experiments. (I,J) Immunostaining of Rab11+ endosomes in fully‐differentiated control and Ap1m2mut enteroids. (J) Quantification of Rab11 apical/cytoplasmic ratio. n = 9–17 enteroids per condition per experiment, from 4 independent technical replicates from 3 biological samples (coloured dots), Mann–Whitney test. On histograms, the total number of enteroids analysed is indicated in brackets.

2.2. AP‐1B Deficiency Leads to Secretory Lineage Differentiation Defects

Besides epithelial polarity defects, we unveiled that Ap1m2 mutations led to severe organisation defects within the epithelial monolayer. Indeed, Olfm4+ ISCs and Lysozyme+ Paneth cells, which are normally confined to the crypt, invaded the villus‐like region of Ap1m2mut enteroids (Figures 2A–D and S2A,B). Furthermore, TEM analysis unravelled the presence of immature/intermediate cells with a mixed zymogen/mucin content, arguing for abnormal cell fate acquisition (Figure 2E,F). Quantifications of these secretory cells in proliferation (crypt and villus‐like regions) or full‐differentiation (villus‐like region only) medium confirmed the presence of Paneth cells in the villus‐like region upon Ap1m2 mutations, and showed that the intermediate cells were present in both conditions (Figure 2G). These strong defects of stem/progenitor zone localisation and secretory lineage differentiation observed in Ap1m2mut enteroids might arise from an imbalance between Notch and Wnt signals, which are crucial in determining the fate of secretory progenitors (Beumer and Clevers 2021). Indeed, membrane targeting of Notch and Wnt receptor has been shown to rely on the AP‐1 complex (Benhra et al. 2011; Wieffer et al. 2013), and inhibition of Notch signalling also induced intermediate cells expressing both Paneth and goblet signatures (Vandussen et al. 2012). Although the expression of Notch and its ligands, as well as that of its major target, Hes1, were not significantly affected by Ap1m2 mutations (Figure S2C), this signalling cascade is based on direct cell–cell contacts that were dramatically disturbed in Ap1m2mut enteroids (Figure 2E and see below), which might impede it. Additionally, defective localisation of EphB2 in Ap1m2mut enteroids (Figure 1H) might reflect an impaired EphB‐EphrinB signalling, which also plays a pivotal function in restricting the localisation of Paneth cells in the crypts (Batlle et al. 2002). Overall, these results showed that AP‐1B controls intestinal cell differentiation both at the subcellular level, through apical–basal polarity establishment, and at the tissular level, through secretory fate acquisition and cell positioning. Interestingly, the rare mixed‐secretory (intermediate) phenotype, together with the invasion of the villus by Paneth cells, was also observed upon Cdc42 KO in mouse (Melendez et al. 2013). Although we could not test the direct effect of Cdc42 downregulation in our system as fully formed Cdc42‐depleted organoids could not be obtained, these data strongly favour a direct involvement of AP‐1B‐mediated polarity control in the differentiation and spatial organisation of the intestinal cells.

FIGURE 2.

FIGURE 2

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AP‐1B deficiency causes secretory lineage differentiation defects. (A–D) Immunostaining of Olfm4 (ISCs) and Lysozyme (Paneth cells) in control and Ap1m2mut enteroids. (A,B) Representative images where crypts and villus‐like regions are delineated in white and magenta, respectively. (C,D) % of enteroids displaying Olmf4+ cells (7–11 enteroids per condition per experiment from 4 independent experiments (dots), unpaired t‐test) or Lysozyme+ cells (n = 6–10 enteroids per condition per experiment from 5 independent experiments (dots), Welch's t test) outside the crypts, respectively. (E–G) Secretory cells were observed by TEM in proliferating (E) and fully‐differentiated (E,F) enteroids, showing Paneth (arrow), goblet (filled arrowheads) and intermediate cells (empty arrowheads). (F) Mixed zymogen/mucin content of intermediate cells at higher magnification. (G) Quantification of the number of Paneth, goblet and intermediate cells from TEM images. Each dot shows a TEM grid (n = 2–4 grid/enteroid from 3–6 enteroids/condition), Mann‐Whitney test. On histograms, the total number of enteroids analysed is indicated in brackets. n.s., non‐significant.

2.3. Exaggeration of β‐catenin Signalling Upon Ap1m2 Silencing may Require a Complete Niche

Organismal Ap1m2 KO in mouse was previously shown to lead to crypt hyperplasia, associated with a cytoplasmic accumulation of E‐cadherin and a nuclear translocation of β‐catenin that ultimately induced proliferation genes (Hase et al. 2013). Using the same general proliferation marker Ki67 (Gerdes et al. 1983; Miller et al. 2018) as in Ap1m2 KO mice (Hase et al. 2013), we observed a similar hyperproliferation in enteroids upon Ap1m2 mutations, which led to a stacking of thinner cells (Figures 3A–C and S3).

FIGURE 3.

FIGURE 3

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The Wnt/β‐catenin pathway is not overactivated in Ap1m2mut enteroids. (A,B) Immunostaining of Ki67 in control and Ap1m2mut enteroids. (B) Quantification of Ki67/DAPI signal. n = 8–26 enteroids per condition per experiment (coloured dots), from 10 independent experiments, Mann–Whitney test. (C) Colorised TEM images of control and Ap1m2mut cell monolayers. (D) TEM analysis of cellular junctions in fully‐differentiated control and Ap1m2mut enteroids. D, desmosome; AJ, adherens junctions; TJ, tight junctions. White arrowheads point to holes between cells. (E) Immunostaining of E‐Cadherin in fully‐differentiated control and Ap1m2mut enteroids. (F) Immunostaining of β‐catenin in the crypts of proliferating control and Ap1m2mut enteroids. Nuclei were stained with DAPI. (G) Quantification of the nuclear/cytoplasmic ratio of β‐catenin in control (507 cells), control + CHIR99021 (CHIR, 20 µM, 3 days) (504 cells) and Ap1m2mut (524 cells) enteroids, from 3 independent experiments, Kruskal–Wallis test. (H) Analysis of β‐catenin target genes transcripts in control, control + CHIR99021 (20 µM, 3 days) and Ap1m2mut enteroids by RT‐qPCR, n = 4 independent experiments, Mann–Whitney test. On histograms, the total number of enteroids analysed is indicated in brackets. n.s., non‐significant.

However, while disorganised intercellular junctions were also observed in Ap1m2mut enteroids, with diffuse and darker tight junctions as well as many holes between cells (Figure 3C–D), we did not observe the previously reported cytoplasmic punctate accumulation of E‐Cadherin in Ap1m2mut enteroids (Figure 3E) (Hase et al. 2013). In order to assess the activation of the β‐catenin pathway, its nuclear localisation and the expression of two of its well‐known transcriptional targets were quantified. As a positive control, enteroids were treated with the GSK3β inhibitor CHIR99021, which prevents β‐catenin targeting to the proteasome. Under these conditions, neither CHIR99021 treatment nor Ap1m2 mutations caused a substantial accumulation of β‐catenin in the nucleus (Figure 3F–G). The slightly higher nuclear accumulation of β‐catenin upon CHIR99021 treatment was nonetheless sufficient to trigger its transcriptional activity, contrary to Ap1m2 crispants, which did not (Figure 3G–H). A full KO of Ap1m2 rather than mutations that do not completely silence it may indeed be required for an overactivation of the Wnt/β‐catenin pathway. However, considering that Wnt/β‐catenin pathway activation relies on agonists released both by Paneth and stromal cells (Beumer and Clevers 2021), our observation raised the possibility that overactivation of this signalling in Ap1m2 null mice may rely on mesenchymal signals. Indeed, these signals are essential for small intestine proliferation and homeostasis in the context of the organ (Valenta et al. 2016), but, except for the Wnt agonist R‐spondin, are absent in the enteroid culture medium. Hence, these results also suggested that other proliferation pathway(s) might be activated upon Ap1m2 mutations.

2.4. AP‐1B Deficiency Activates an mTOR/YAP‐Dependent Proliferation Pathway

To search for putative complementary proliferation pathways activated upon AP‐1B deficiency, we first analysed publicly available gene expression profiling data from Ap1m2 null mice (Gene Expression Omnibus Database #GSE22020) (Hase et al. 2013). Interestingly, this suggested that mechanistic target of rapamycin (mTOR), whose activity has been shown to partly depend on AP‐1 in C. elegans (Zhu et al. 2015), and yes‐associated protein (YAP) proliferation pathways might be engaged. To test the involvement of mTOR signalling in the hyperproliferation of Ap1m2mut enteroids, they were treated with the selective inhibitor of mTOR, rapamycin (Hay and Sonenberg 2004). Remarkably, rapamycin treatment rescued the proliferation of Ap1m2mut enteroids to control levels without significantly influencing the proliferation of control enteroids (Figure 4A–B), strongly suggesting that the mTOR pathway is specifically induced by Ap1m2 mutations.

FIGURE 4.

FIGURE 4

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Ap1m2 mutations activate an mTOR and YAP‐dependent proliferation pathway. (A,B) Quantification of Ki67/DAPI ratio in control and Ap1m2mut enteroids treated or not with rapamycin (1 µM, 3 days). (A) n = 7–10 enteroids per condition per experiment (coloured dots), from 6 independent experiments, Kruskal–Wallis test. (B) n = 8–10 enteroids per condition per experiment (coloured dots), from 3 independent experiments, Mann–Whitney test. Control samples were part of the controls in (A). (C) Immunofluorescence staining of YAP and Ki67 in the crypts of Ap1m2mut enteroid slices. Nuclei were stained with DAPI, and arrows show nuclei with high levels of YAP and Ki67. (D) Quantification of nuclear YAP signal intensity in control and Ap1m2mut crypts, n = 10–16 enteroids per condition per experiment (coloured dots), from 3 independent experiments, Welch's t test. (E) RT‐qPCR analysis of YAP pathway transcripts in control and Ap1m2mut enteroids. n = 5–7 independent experiments, Mann–Whitney test. (F) Maximum intensity projection of a z‐stack of control enteroids stained for YAP and nuclei (DAPI), the dotted lines show the ROIs that were selected for quantification of YAP signal in the whole crypt volume. (G) Quantification of nuclear YAP in the whole crypts of control and Ap1m2mut enteroids, treated or not with rapamycin (1 µM, 3 days). n = 5–10 enteroids per condition per experiment (coloured dots), from 4 independent experiments, Kruskal–Wallis test. (H) Quantification of the % of proliferating enteroids displaying Paneth cells in the villus‐like region in control and Ap1m2mut enteroids, treated or not with rapamycin. n = 5–10 enteroids per condition per experiment, from 3 independent experiments, Mann–Whitney test. On histograms, the total number of enteroids analysed is indicated in brackets. n.s., non‐significant.

Next, we tested the involvement of YAP in the proliferation of Ap1m2mut enteroids. Cell proliferation is triggered when the Hippo pathway is inactive, YAP being translocated to the nucleus upon release from phosphorylation‐dependent degradation, allowing the induction of target genes by means of its interaction with TEAD1–4 transcription factors (Li et al. 2025). First, the localisation of YAP in both enteroid populations showed that part of the crypt cells that express high levels of nuclear YAP were also strongly positive for Ki67 (Figure 4C), in line with the established function of YAP in ISC self‐renewal (Deng et al. 2022). Of note, quantification of nuclear YAP in the crypts of Ap1m2mut enteroids showed a 1.2–1.6‐fold increase compared to control, indicating that the YAP pathway is more active upon AP‐1B inhibition (Figure 4D, G). To confirm this hypothesis, the mRNA levels of various YAP target genes were examined. We found that two targets of the YAP pathway were significantly up‐ and down‐regulated by AP‐1B deficiency, respectively: connective tissue growth factor (CTGF) and peripheral myelin protein 22 (PMP22) (Figure 4E). PMP22 is found at cell junctions in the colon and seems to be downregulated in colon carcinoma (Notterpek et al. 2001; Zhang et al. 2022b), but, to our knowledge, has not been directly studied in the context of gut cell renewal. Conversely, CTGF has been shown to be upregulated during intestine regeneration (Guillermin et al. 2021; Kim et al. 2017) and is thus a good candidate for contributing to Ap1m2 mutations‐induced overproliferation downstream of YAP. Collectively, these results indicate that Ap1m2 mutations in intestinal epithelia trigger a tissue cell‐autonomous proliferation pathway that involves mTOR and YAP. As the hierarchy between these two signalling pathways seems to be context‐specific (Honda et al. 2023), epistasis was tested by quantifying YAP nuclear localisation in the whole crypts of Ap1m2mut enteroids treated or not with rapamycin (Figure 4F). We observed a rescue of nuclear YAP localisation to control levels, demonstrating that YAP acts downstream of mTOR in Ap1m2mut enteroids (Figure 4G). Interestingly, silencing of Cdc42 or Rab11, whose sorting relies on AP‐1B, also induced a YAP‐dependent intestinal cell hyperplasia (D'Agostino et al. 2019; Goswami et al. 2021; Zhang et al. 2022a). However, in Cdc42‐KO intestinal cells, YAP was upstream of mTOR (Zhang et al. 2022a), which suggests that different proliferation pathways are associated with the mislocalisation or the silencing of Cdc42 and Rab11.

In conclusion, we have expanded the functional repertoire of AP‐1B in gut homeostasis. We showed here that AP‐1B, in addition to enterocyte apical–basal polarity, controls secretory lineage fate acquisition and positioning along the crypt‐villus axis. In addition, we propose that Ap1m2 downregulation triggers two different proliferation pathways: a mesenchyme‐dependent Wnt/β‐catenin pathway (Hase et al. 2013) and a tissue‐autonomous pathway that involves mTOR and YAP. Since YAP and mTOR pathways have also been involved in intestinal epithelia differentiation (Imajo et al. 2015; Zhou et al. 2015), their alteration may contribute to the differentiation defects found in Ap1m2mut enteroids. However, because rapamycin failed to rescue Paneth cells defects (Figure 4H), our data suggest that AP‐1B might control intestinal cells proliferation and differentiation independently. Moreover, it has been proposed that mTOR and YAP pathways are specifically involved in intestinal regeneration upon injury (Gregorieff et al. 2015; Sampson et al. 2016). It is thus possible that the activation of these pathways originated from a regenerative mechanism aiming at compensating for the major intestinal monolayer defects observed in Ap1m2mut enteroids. Further investigations are thus needed to refine the mechanisms by which AP‐1B controls intestinal cell differentiation and proliferation. Notably, analyses at the scale of individual cells (i.e., scRNA‐seq) may allow for better assessment of the potency of mTOR and YAP pathways activation in dividing cells, to highlight more YAP transcriptional targets, and to know whether other cells than Lgr5+ ISCs are involved in the exaggerated proliferation upon AP‐1B complex disruption.

3. Material and Methods

3.1. Chemicals

Rapamycin was purchased at APExBIO (A8167), CHIR99021 was from Ozyme (STE04‐0004).

3.2. Enteroid Culture

Mouse intestinal crypts were cultured in drops of Cultrex reduced growth factor basement membrane extract type 2 (BME2, 3533‐010‐02, Bio‐techne) seeded on 12‐well Greiner Cell STAR multiwell low retention plates with ENR medium (Advanced DMEM/F12 (12634028), 10 mM Hepes (15630056), 1% Glutamax (35050‐038), 1% Penicillin/Streptomycin, 1X B27 supplement (17504‐044) (all from Life technologies), 1.25 mM N‐acetylcysteine (A9165, Sigma–Aldrich), 50 ng/mL hEGF (AF‐100‐15, Peprotech), supplemented with 5% R‐Spondin1 and 10% Noggin conditioned media produced as described earlier (Mosa et al. 2018; Sato et al. 2011). Medium was changed every 2–3 days, and enteroids were weekly passaged by mechanical disruption. When indicated, full differentiation was achieved by a treatment for 6 days with 1 mM valproic acid (P4543, Sigma–Aldrich), with the addition of 2.5 µg/mL inhibitor of WNT production‐2 (IWP‐2, I0536, Sigma–Aldrich) for the last 3 days. In experiments, rapamycin and CHIR99021 were added to the culture medium for the last 3 days of culture. Absence of mycoplasma was checked regularly.

3.3. Conditional Gene Mutation by CRISPR‐Cas9

Inducible Cas9 nuclease‐expressing ISC (derived from a wild‐type male mouse), a kind gift from Delphine Delacour (IBDM, Marseille, France) were transduced with EditR pre‐designed sgRNAs lentiviral particles (Horizon discovery, Cambridge, UK) as described earlier (Bidaud‐Meynard et al. 2024; Saleh et al. 2023). Transduced enteroids were selected with 5 µg/mL Blasticidin (Sigma 15205)/3 µg/mL Puromycin (A1113803, Gibco). Gene mutation was induced 4 days after passage during 6 days with 400 ng/mL doxycycline (D9891, Sigma–Aldrich). The % of indels was calculated using ICE software (Conant et al. 2022) after Sanger sequencing of enteroids' genomic DNA. Primers and sgRNAs are listed in Table S1.

3.4. Analysis of Gene Expression by RT‐qPCR

Total RNA was isolated from BME2‐extracted enteroids using the Nucleospin RNA Plus kit (Macherey–Nalgen, 740984). cDNAs were obtained by reverse transcription using oligo(dT) (Promega, C1101) and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, 18080093) from identical mRNA amounts. Gene expression was assessed by quantitative real‐time PCR performed on a QuantStudio 7 Flex system (Thermo Fisher Scientific), using the Power SYBR Green PCR master mix (Thermo Fisher Scientific, 4367659). Expression of the housekeeping gene Hprt was used as an internal control for normalisation. Primers used are indicated in Table S1.

3.5. Transmission Electron Microscopy

Enteroids were collected from BME2 and fixed in Trump's fixative at 4°C. Enhanced chemical fixation was performed in 4% paraformaldehyde/2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) overnight at 4°C. Then, enteroids were incubated successively in 1% OsO4 for 1.5 h and 2% uranyl acetate for 1.5 h, at RT. Enteroids were then dehydrated through graded ethanol solutions, cleared in acetone, and flat‐embedded in Epon‐Araldite mix (EMS hard formula) using adhesive frames (11560294 GENE‐FRAME 65 µL, Thermo Fisher Scientific). Ultrathin 70 nm sections were collected on formvar‐coated slot grids (FCF2010‐CU, EMS). TEM grids were observed using a JEM‐1400 TEM (JEOL) operated at 120 kV, equipped with a Gatan Orius SC1000 camera piloted by the Digital Micrograph 3.5 software.

3.6. Immunostaining

Enteroids were collected from BME2 and fixed in 2% PFA/PBS1X overnight at 4°C. For immunostaining on enteroid slices, 4 µm paraffin bloc sections were mounted on slides before deparaffination in Xylene followed by decreasing ethanol baths. After rehydration, antigen retrieval was performed with Tris‐EDTA, pH8 (98°C, 40 min). Then, both whole and sliced enteroids were permeabilized in PBS1X, 0.1% Tween20, 0.2% Triton, blocked in PBS1X, 0.1% Tween20, 2% foetal bovine serum and incubated overnight at 4°C in blocking solution with primary antibodies (Supplementary Table 2). After incubation with adequate fluorescent secondary antibodies (Jackson ImmunoResearch) during 1 h at room temperature, they were mounted in ProLong Gold Antifade reagent (P36941, Life Technologies). Imaging was performed on a Zeiss LSM880‐Airyscan (Oberkochen, Germany) equipped with a 63X, 1.4 NA objective (Zen Black software) and images were analysed using Fiji.

3.7. Image Quantification

Nuclear localisation of Ki67 and YAP on all the optical sections of whole enteroids and cropped crypts, respectively, was quantified by measuring the signal on a nuclear overlay obtained after nuclear segmentation using the DAPI channel in Fiji. This signal was then divided by the signal of the same overlay in the DAPI channel to correct for intensity differences due to the varying distance between the cells and objective. To measure YAP nuclear localisation on sliced enteroids, the images were segmented on the DAPI channel and the signal was measured on the same ROI in the YAP channel. To measure P‐ezrin lateral+cytoplasmic/cytoplasmic ratio, the signal of a segmented line crossing the equator of the cells was divided by the whole cell signal after threshold‐based removal of the membranous P‐ezrin signal. The apical/cytoplasmic ratio of Rab11 was calculated by measuring the signal intensity of a segmented line covering the apical membrane and measuring the signal intensity of an identical ROI in the underlying cytoplasm. To measure the nuclear/cytoplasmic ratio of β‐catenin on sliced enteroids, labelled images were obtained using Cellpose v3.1.0 segmentation in FIJI with the BIOP plugin (Stringer et al. 2021). The pre‐trained model cyto2 was used for both nuclei and intracellular segmentation. Resulting labels were then eroded by 6 pixels using the « LabelToROIs » plugin (Waisman et al. 2021). Next, nuclei ROIs were used to remove the nuclei area from intracellular labels using the fill function to generate the cytoplasmic ROIs, and the nuclear and cytoplasmic signals were measured. Cell width was calculated by dividing the length of a line crossing the equator of the cells by the number of nuclei (DAPI channel) on the same optical section.

3.8. Statistical Analysis and Data Presentation

Statistical analysis and graphic representations were performed with Graphpad Prism 11. Histograms show the mean and standard deviation (black bars). Gaussian distribution and homoscedasticity of data were verified by the Shapiro–Wilk test and the F‐test, respectively. A two‐tailed non‐parametric Mann–Whitney test was performed when at least part of the datasets did not pass the normality test or when data were normalised to control. For normalised datasets, an exact p value, which takes into account ties among values, was computed by Prism software. The tests used for the analysis of the statistical significance of data are indicated in the legends. Images shown are representative of at least 3 independent experiments.

Funding

This work was supported by La Ligue contre le cancer Grand Ouest (35/85 and 29/35/85), by the Agence Nationale de la Recherche (ANR‐23‐CE13‐0003), as well as by institutional funding from IGDR, the Centre National de la Recherche Scientifique and the Université de Rennes.

Conflicts of Interest

The authors declare no conflicts of interest. LLM/AI use in this manuscript was restricted to image quantification.

Supporting information

Supporting File: boc70076‐sup‐0001‐SuppMat.pdf.

BOC-118-e70076-s001.pdf (1.3MB, pdf)

Acknowledgements

We thank Hans Clevers, Calvin Kuo and Delphine Delacour for Noggin, R‐Spondin1 and CAS9‐expressing cells, respectively. We are grateful to Gerard Benoit, Christophe Hitte, and Justine Viet for their help with data analysis, to Guillaume Halet for critical reading of the manuscript, and to members of the Michaux lab for helpful discussions. Paraffin bloc sections and imaging were performed at the Histo Pathology High Precision (H2P2) and the Microscopy Rennes Imaging Centre (MRiC) facilities (BIOSIT, Biogenouest), members of the national infrastructure France‐BioImaging supported by the French National Research Agency (ANR‐24‐INBS‐0005 FBI BIOGEN). Image quantification tools were developed with the help of Thierry Pecot from the Facility for Artificial Intelligence and Image Analysis (FAIIA), Biosit UAR 3480 CNRS‐US 18 INSERM, Rennes.

Open access publication funding provided by COUPERIN CY26.

Duclos, M. , Bourdais A., Nicolle O., Helpiquet A., Michaux G., and Bidaud‐Meynard A.. 2026. “AP‐1B Controls Several Levels of Cell Proliferation and Differentiation in Mouse Enteroids.” Biology of the Cell 118, no. 8: e70076. 10.1111/boc.70076

Grégoire Michaux and Aurélien Bidaud‐Meynard should be considered joint senior authors.

Contributor Information

Grégoire Michaux, Email: gregoire.michaux@univ-rennes.fr.

Aurélien Bidaud‐Meynard, Email: pierre-aurelien.bidaud@univ-rennes.fr.

Data Availability Statement

Data available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File: boc70076‐sup‐0001‐SuppMat.pdf.

BOC-118-e70076-s001.pdf (1.3MB, pdf)

Data Availability Statement

Data available on request.

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