MEX3A regulates Lgr5 + stem cell maintenance in the developing intestinal epithelium
2020; Springer Nature; Volume: 21; Issue: 4 Linguagem: Inglês
10.15252/embr.201948938
ISSN1469-3178
AutoresBruno Pereira, Ana Luísa Amaral, Alexandre Dias, Nuno Mendes, Vanesa Muncan, Ana R Silva, Chantal Thibert, A Radu, Leonor David, Valdemar Máximo, Gijs R. van den Brink, Marc Billaud, Raquel Almeida,
Tópico(s)Digestive system and related health
ResumoArticle13 February 2020Open Access Transparent process MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium Bruno Pereira Corresponding Author Bruno Pereira [email protected] orcid.org/0000-0003-2460-6720 i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Ana L Amaral Ana L Amaral i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Alexandre Dias Alexandre Dias i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Nuno Mendes Nuno Mendes i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Vanesa Muncan Vanesa Muncan Department of Gastroenterology and Hepatology, Amsterdam UMC, Tytgat Institute, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Ana R Silva Ana R Silva i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Chantal Thibert Chantal Thibert orcid.org/0000-0002-1516-5671 Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, University Grenoble Alpes, Grenoble, France Search for more papers by this author Anca G Radu Anca G Radu Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, University Grenoble Alpes, Grenoble, France Search for more papers by this author Leonor David Leonor David i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal FMUP-Faculty of Medicine, University of Porto, Porto, Portugal Search for more papers by this author Valdemar Máximo Valdemar Máximo i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal FMUP-Faculty of Medicine, University of Porto, Porto, Portugal Search for more papers by this author Gijs R van den Brink Gijs R van den Brink Department of Gastroenterology and Hepatology, Amsterdam UMC, Tytgat Institute, University of Amsterdam, Amsterdam, The Netherlands Medicines Research Center, GSK, Stevenage, UK Search for more papers by this author Marc Billaud Marc Billaud Clinical and Experimental Model of Lymphomagenesis, INSERM U1052, CNRS UMR5286, Centre Léon Bérard, Université Claude Bernard Lyon 1, Centre de Recherche en Cancérologie de Lyon, Lyon, France Search for more papers by this author Raquel Almeida Corresponding Author Raquel Almeida [email protected] orcid.org/0000-0002-5622-1633 i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal FMUP-Faculty of Medicine, University of Porto, Porto, Portugal Biology Department, Faculty of Sciences, University of Porto, Porto, Portugal Search for more papers by this author Bruno Pereira Corresponding Author Bruno Pereira [email protected] orcid.org/0000-0003-2460-6720 i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Ana L Amaral Ana L Amaral i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Alexandre Dias Alexandre Dias i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Nuno Mendes Nuno Mendes i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Vanesa Muncan Vanesa Muncan Department of Gastroenterology and Hepatology, Amsterdam UMC, Tytgat Institute, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Ana R Silva Ana R Silva i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Search for more papers by this author Chantal Thibert Chantal Thibert orcid.org/0000-0002-1516-5671 Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, University Grenoble Alpes, Grenoble, France Search for more papers by this author Anca G Radu Anca G Radu Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, University Grenoble Alpes, Grenoble, France Search for more papers by this author Leonor David Leonor David i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal FMUP-Faculty of Medicine, University of Porto, Porto, Portugal Search for more papers by this author Valdemar Máximo Valdemar Máximo i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal FMUP-Faculty of Medicine, University of Porto, Porto, Portugal Search for more papers by this author Gijs R van den Brink Gijs R van den Brink Department of Gastroenterology and Hepatology, Amsterdam UMC, Tytgat Institute, University of Amsterdam, Amsterdam, The Netherlands Medicines Research Center, GSK, Stevenage, UK Search for more papers by this author Marc Billaud Marc Billaud Clinical and Experimental Model of Lymphomagenesis, INSERM U1052, CNRS UMR5286, Centre Léon Bérard, Université Claude Bernard Lyon 1, Centre de Recherche en Cancérologie de Lyon, Lyon, France Search for more papers by this author Raquel Almeida Corresponding Author Raquel Almeida [email protected] orcid.org/0000-0002-5622-1633 i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal FMUP-Faculty of Medicine, University of Porto, Porto, Portugal Biology Department, Faculty of Sciences, University of Porto, Porto, Portugal Search for more papers by this author Author Information Bruno Pereira *,1,2, Ana L Amaral1,2,‡, Alexandre Dias1,2,‡, Nuno Mendes1,2, Vanesa Muncan3, Ana R Silva1,2, Chantal Thibert4, Anca G Radu4, Leonor David1,2,5, Valdemar Máximo1,2,5, Gijs R van den Brink3,6, Marc Billaud7 and Raquel Almeida *,1,2,5,8 1i3S - Institute for Research and Innovation in Health (Instituto de Investigação e Inovação em Saúde), University of Porto, Porto, Portugal 2IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal 3Department of Gastroenterology and Hepatology, Amsterdam UMC, Tytgat Institute, University of Amsterdam, Amsterdam, The Netherlands 4Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, University Grenoble Alpes, Grenoble, France 5FMUP-Faculty of Medicine, University of Porto, Porto, Portugal 6Medicines Research Center, GSK, Stevenage, UK 7Clinical and Experimental Model of Lymphomagenesis, INSERM U1052, CNRS UMR5286, Centre Léon Bérard, Université Claude Bernard Lyon 1, Centre de Recherche en Cancérologie de Lyon, Lyon, France 8Biology Department, Faculty of Sciences, University of Porto, Porto, Portugal ‡These authors contributed equally to this work *Corresponding author. Tel: +351 2260 74900; E-mail: [email protected] *Corresponding author. Tel: +351 2260 74900; E-mail: [email protected]p.pt EMBO Reports (2020)21:e48938https://doi.org/10.15252/embr.201948938 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Intestinal stem cells (ISCs) fuel the lifelong self-renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA-binding protein is crucial for the maintenance of the Lgr5+ ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator-activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5+ stem cell signature. Furthermore, we identify PPARγ activity as a molecular intermediate of MEX3A-mediated regulation. We also show that high PPARγ signalling impairs Lgr5+ ISC function, thus uncovering a new layer of post-transcriptional regulation that critically contributes to intestinal homeostasis. Synopsis The RNA-binding protein MEX3A is expressed at the base of intestinal crypts and is critical for the maintenance of Lgr5+ ISCs during postnatal development. MEX3A regulates the PPARγ pathway, thereby modulating ISC identity and tissue homeostasis. Mex3a−/− mice exhibit growth retardation, postnatal mortality, and a severe impairment of intestinal crypt development. Crypts of Mex3a knockout mice exhibit loss of Lgr5+ intestinal stem cells and disruption of epithelial turnover. Mex3a deletion leads to the aberrant activation of the PPARγ signalling pathway in intestinal crypts. High PPARγ signalling impairs Lgr5+ stem cell function in intestinal organoids. Introduction Stem cells ensure homeostasis throughout life, and the intestinal tract is the organ that best displays this extraordinary ability. It is the fastest self-renewing tissue in mammals, as epithelial turnover takes about 3–5 days to be completed 1. Intestinal stem cells (ISCs), located in mucosal invaginations known as crypts of Lieberkühn, control this process. ISCs generate proliferating transit-amplifying (TA) cells that after several divisions commit into differentiated cell types. These mainly include nutrient-absorbing enterocytes, mucous-producing goblet cells, hormone-secreting enteroendocrine cells and chemosensing tuft cells; all located along luminal protrusions called villi. Paneth cells, which play a role in innate immunity by synthesizing antimicrobial peptides, are unique because they are the only differentiated cell type that stays in the crypt with a long residence time of 6–8 weeks. The location and nature of ISCs remains a matter of intense debate. Expression profile and lineage tracing experiments performed over the last decade have led to the identification of at least two distinct ISC populations: crypt base columnar (CBC) cells and +4 cells. CBC cells, wedged between Paneth cells at the base of the crypt, are actively cycling and sensitive to radiation-induced damage. The Wnt/β-catenin pathway target gene Lgr5 is one of the most reliable CBC markers 2, and single Lgr5+ cells originate organoids or "mini-gut"-like structures ex vivo comprising all intestinal cell types 3. Additional markers for CBC stem cells include Olfm4 and Ascl2 45. The +4 cells, located on average 4 positions above the crypt bottom and over the uppermost Paneth cell, are relatively quiescent and resistant to radiation, expressing markers such as Bmi1, Hopx, Lrig1 and Tert 678910. These populations are not hard-wired, and intricate patterns of interconversion seem to exist between them, both under homeostasis and in response to injury 10111213. Moreover, it has been shown that Dll1+ 14, Atoh1+ 151617 or Prox1+ 18 secretory progenitor cells, Alpi+ enterocyte precursors 19 and even subsets of committed Lyz1+ Paneth cells 2021 can all present stem cell activity, illustrating the level of cellular plasticity in the gut. Most studies focusing on ISC biology have been performed using adult animal models, when intestinal homeostasis is stably established, whereas little is known about one of the most active phases of this cellular population, which is the postnatal period between birth and weaning 22. Newborn mice possess an immature epithelium that contains villi formed in late embryogenesis but lacks crypts. Instead, a small number of Lgr5+ progenitors are restricted to basal regions between villi called intervillus domains 2324. The foetal LGR5 progeny is, by itself, insufficient to sustain intestinal growth during initial development 25, suggesting the presence of Lgr5− precursors in the earliest phases of postnatal development. To ensure proper epithelial cell renewal, the functional crypt-villus axis must develop rapidly after birth, which is accomplished by an initial expansion of the entire stem cell pool via symmetric divisions, followed by a sharp transition to TA cell production via asymmetric divisions 26. In this phase, the intestinal epithelium is not in a steady-state condition, as in the adult counterpart, because cell production rate necessarily exceeds cell loss. Concomitant with these structural alterations, the epithelium undergoes major biochemical changes to support the dietary adaptation associated with the suckling-to-weaning transition 2728. The exact mechanisms involved in initiating postnatal expansion of ISCs and controlling its timing are unknown, but probably involve an intrinsic genetic programme and microenvironmental factors (e.g. circulating hormones, diet, microbiota), as well as molecular determinants mediating communication between both. It is becoming clear that RNA-binding proteins (RBPs) and post-transcriptional mechanisms underpin stem cell fate decisions in response to different stimuli 2930. In this regard, we have been studying the evolutionarily conserved MEX-3 family of RBPs. Vertebrates have four homologous genes designated MEX3A to MEX3D encoding related proteins with two K Homology (KH) domains that provide RNA-binding capacity 31, and a Really Interesting New Gene (RING) C-terminal domain, which possibly mediates E3 ubiquitin ligase activity 32. The different MEX-3 members are post-transcriptional regulators involved in embryonic patterning 33, pluripotency 34, fertility 35, immune responses 36, metabolism 37 and cancer 38. Our previous work demonstrated that MEX3A overexpression is associated with stemness features in gastrointestinal cancer cell lines, including higher expression of the ISC markers LGR5, BMI1 and MSI1 39. In agreement, Mex3a mRNA is part of the Lgr5+ signature 40, and its expression upregulated in a mouse model overexpressing MSI1 in the intestinal epithelium 41. Recently, Mex3a expression was observed in a subset of Lgr5+ cells around the crypt +3/+4 cell position 42. However, none of the previous studies has functionally addressed if MEX3A is essential in the context of ISCs. In the present study, through the detailed characterization of a novel mouse model with a Mex3a deletion, we show for the first time that MEX3A is critical for the in vivo maintenance of the Lgr5+ ISC pool. Mex3a null mice exhibit growth retardation and postnatal mortality due to impaired epithelial turnover, underlined by a dramatic decrease in Lgr5+ ISCs and TA cells. Additionally, we provide evidence that Mex3a deletion leads to the aberrant activation of the peroxisome proliferator-activated receptor (PPAR) signalling pathway and establish PPARγ signalling as a molecular intermediate of MEX3A-mediated regulation. Our data uncover a new regulatory mechanism in ISCs of the developing gut with implications for intestinal homeostasis. Results Characterization of Mex3a expression pattern in murine tissues We started by examining the Mex3a expression pattern among major organs in the mouse during postnatal development. By in situ hybridization (ISH), we determined that Mex3a mRNA was highly expressed in the thymus, moderately expressed in the brain and gut, lowly expressed in the stomach and skin, and absent from the heart, liver and lung (Fig EV1). In the intestinal tract, Mex3a transcripts were concentrated at the base of the small intestine and colonic crypts (Fig EV1, small intestine and colon inserts). In the skin, Mex3a mRNA was present in hair follicle-related structures only (Fig EV1, skin insert). The precise compartmentalization of Mex3a expression in stem cell niches of two of the most rapidly self-renewing mammalian organs, intestine and skin, suggested an in vivo function for MEX3A in stem cell biology. Click here to expand this figure. Figure EV1. Characterization of Mex3a expression pattern in murine tissuesH&E staining and Mex3a mRNA ISH in serial sections of different mouse organs at postnatal day 17. Each punctuate red dot in the ISH panels represents a hybridization event with a single Mex3a mRNA molecule. Inserts depict high magnification of the boxed areas. The diffuse signals observed in the liver are the result of non-specific staining. Scale bars, 50 μm. Download figure Download PowerPoint Mex3a null mice exhibit growth retardation and postnatal mortality To address the physiological role of Mex3a, we characterized mice with a targeted intragenic deletion of the Mex3a locus coding sequence, developed under the framework of the INFRAFRONTIER-I3 European Research Infrastructure 43. The initial deletion cassette consisted of a LacZ reporter cDNA followed by a floxed promoter-driven neomycin (Neo) resistance gene (Fig 1A). The Mex3atm1(KOMP)Vlcg strain was generated and crossed with the epiblast-specific Meox2+/Cre deleter strain for removal of the Neo gene, giving rise to Mex3a+/− heterozygous mice. Heterozygous breeding schemes were set up and Mex3a−/− homozygous mutant animals were born, although not at the expected Mendelian frequency (13% versus 25% expected, Fig 1B), indicating the occurrence of embryonic lethality. Figure 1. Mex3a knockout mice exhibit smaller size and postnatal lethality Scheme of the targeting vector for intragenic deletion of the mouse Mex3a gene. The insertion of the Velocigene cassette ZEN-Ub1 created a deletion of 1,125 bp in exon 2 of the Mex3a locus. Representative images of the size of Mex3a mutant mice and control littermates at postnatal day (P)15. Scale bar, 1 cm. Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue (right panels). Scale bars, 50 μm. The offspring number (n) and observed genotype frequencies (%) resulting from heterozygous crosses are indicated below. Absolute weight of Mex3a KO mice and control littermates at different ages. Data are represented in a box-and-whisker plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one animal (WT: P1, n = 15; P8, n = 36; P15, n = 43; P22, n = 14; Adult, n = 14; Mex3a+/−: P1, n = 24; P8, n = 90; P15, n = 90; P22, n = 37; Adult, n = 21; Mex3a−/−: P1, n = 9; P8, n = 24; P15, n = 26; P22, n = 14; Adult, n = 8) ***P = 0.0003, ****P < 0.0001, two-way ANOVA test. Kaplan–Meier survival curves for the different Mex3a genotypes (n = 80 for each genotype) ****P < 0.0001, log-rank (Mantel–Cox) test. Download figure Download PowerPoint Mex3a knockout (KO) pups displayed severe growth retardation, presenting smaller size and weight when compared to Mex3a+/− heterozygous and wild-type (WT) siblings (Fig 1B and C). At postnatal day (P)15, Mex3a KO animals had an average weight of 4.00 ± 0.16 g (mean ± standard error, n = 26), compared to 6.57 ± 0.14 g (n = 43) of WT mice, a 39% weight difference (Fig 1C). This trait persists in mutant mice that reach adulthood. Both adult males and females are fertile and seem to have a normal lifespan. However, around 60% of the mutant animals developed a clinical condition characterized by gradual weight loss, signs of dehydration and progressive lethargy, between P15 and P21, eventually culminating in death (Fig 1D). These pups displayed normal suckling behaviour, as judged by the presence of milk in their stomachs (Appendix Fig S1). Macroscopic assessment of major organs indicated no gross morphological abnormalities, with the notable exception of the intestinal tract (Fig 2A and Appendix Fig S1). This subset of Mex3a null mice presented a translucent and air-filled gut tube, particularly evident in the ileum, caecum and colon (Fig 2A). The overlap between the observed phenotype and the occurrence of important events in murine intestinal ontogenesis during this developmental time-window prompted us to focus on the effect of Mex3a deletion specifically in the intestinal epithelium. Figure 2. Mex3a deletion leads to loss of Lgr5+ stem cells and impairs normal intestinal crypt development A. Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice. These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days. Boxed areas depict the distal small intestinal section (ileum) used for subsequent immunohistochemical analyses. B. H&E staining of a Mex3a KO and WT littermate at P19. Inserts depict high magnification of the boxed areas. C, D. Average crypt depth (C) of WT animals (n = 3, > 40 crypts counted per animal) and Mex3a mutants (n = 6, > 20 crypts counted per animal). Also shown is the average villi height (D) of WT animals (n = 3, > 25 villi counted per animal) and Mex3a mutants (n = 6, > 15 villi counted per animal). Data are represented as mean ± standard error between P16 and P18. *P = 0.03, ***P = 0.0006, Student's t-test. E–G. Immunohistochemistry staining for (E) the tuft cell marker DCLK1 (black arrowheads indicate positive cells), (F) the Paneth cell marker LYZ1 and (G) the proliferation marker KI67. H–K. mRNA ISH staining for the stem cell markers (H) Lgr5, (I) Olfm4, (J) Lrig1 and (K) Hopx in ileal sections of Mex3a mutants and littermate controls at P19. Inserts depict high magnification of the boxed areas. L. qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals (n = 3 of each genotype). Data are represented as mean fold-change plus standard error in Mex3a KO mice relatively to WT animals (dashed line). *P = 0.0208, **P < 0.01, ***P = 0.0007, Student's t-test. Data information: Scale bar (A), 1 cm; all other scale bars, 50 μm. Download figure Download PowerPoint Mex3a deletion severely impairs normal intestinal crypt development Histological analysis of haematoxylin and eosin (H&E)-stained paraffin sections of the intestinal tissue of Mex3a KO animals revealed severely altered crypt-villus architecture, particularly in the distal small intestine, with the presence of a reduced number of crypts and of smaller size when compared to littermate or age-matched control animals (Fig 2B). Average crypt depth in Mex3a KO mice was 17.66 ± 1.33 μm compared with 30.05 ± 1.20 μm in WT mice (Fig 2C). Intestinal villi of mutant animals were also shorter, with a mean height of 98.86 ± 6.60 μm compared with 131.50 ± 10.82 μm in control mice (Fig 2D). A similar trend was observed for the proximal small intestine (Appendix Fig S2). To explore the consequences of disrupting the Mex3a gene for intestinal differentiation, we assessed the presence of the main epithelial cell types using specific markers. The goblet and enteroendocrine cell lineages were not significantly modified as determined by alcian blue–periodic acid–Schiff (AB-PAS) reaction (Fig EV2A) and synaptophysin (SYP) staining (Fig EV2B), respectively, with a normal distribution of scattered cells amidst the mucosa. Enterocytes were equally detected along the lining of the villi by expression of villin (VIL1), a structural marker for the apical brush border (Fig EV2C). On the other hand, tuft cells were almost entirely absent from the Mex3a mutant mice intestine as observed with doublecortin-like kinase 1 (DCLK1), a specific tuft cell marker (Fig 2E). Since we previously demonstrated in cancer cell lines that MEX3A post-transcriptionally represses the expression of caudal-type homeobox 2 (CDX2) protein 39, a master regulator of intestinal maturation, we assessed CDX2 expression in the mutant animals. Although not consistently observed, CDX2 levels seemed increased in the incipient crypt compartment of Mex3a KO mice when compared with WT (Fig EV2D). Considering the hypothesis of a premature maturation of the intestinal epithelium, we tested the expression of sucrase-isomaltase (SIS, Fig EV2E) by immunohistochemistry. Normally, this enzyme starts to be expressed during the suckling-to-weaning transition and accompanies the change to solid food, being involved in the digestion of complex carbohydrates 2728. A premature expression of SIS was not detected in Mex3a KO mice at P15. As expected, the suckling period-specific enzyme argininosuccinate synthetase 1 (ASS1), which is involved in arginine synthesis, was broadly present in both mutant and control animals in the same period (Fig EV2F). Click here to expand this figure. Figure EV2. Mex3a mutant mice do not exhibit a premature maturation of the intestinal epithelium A–C. The main differentiated cell types are present in the Mex3a mutant mice intestinal epithelium, namely goblet cells as assessed by (A) alcian blue–periodic acid–Schiff (AB-PAS) reaction, (B) enteroendocrine cells as assessed by Synaptophysin (SYP, black arrowheads) expression and (C) enterocytes as assessed by Villin (VIL1) staining. D. Immunohistochemical staining for the intestinal transcription factor CDX2. E. Immunohistochemistry shows that sucrase-isomaltase (SIS) expression at the brush border is only observed at the suckling-to-weaning transition in the Mex3a mutant mice, as well as in the control. F. Expression of the suckling period-specific enzyme argininosuccinate synthetase 1 (ASS1) is also observed in the correct time-point. Data information: All scale bars, 50 μm. Download figure Download PowerPoint Regarding crypt cell populations, we detected a strong inhibition of Paneth cell expansion as assessed by lysozyme (LYZ1) staining in the Mex3a KO intestine (Fig 2F). We equally detected a significant lower number of proliferating cells identified by KI67 staining in the Mex3a KO intestine (Fig 2G). The average number of KI67+ cells per crypt was 6.56 ± 1.39 compared with 12.60 ± 1.07 in WT mice, a 48% difference (Appendix Fig S3). Collectively, these data demonstrate that MEX3A is not directly involved in the intrinsic mechanisms associated with intestinal maturation but is necessary for normal crypt development. Loss of Lgr5+ stem cells in the small intestine of Mex3a KO mice Given the pronounced atrophy detected at the crypt level, we decided to investigate whether Mex3a deletion influenced ISCs responsible for maintaining small intestine epithelial homeostasis. For that, we performed ISH for the specific CBC cell marker Lgr5 2. Strikingly, we observed a strong reduction in Lgr5 mRNA levels in the intestinal crypts of Mex3a KO animals when compared to WT (Fig 2H). This reduction could be the result of either diminished Lgr5 gene transcription or loss of the Lgr5-expressing ISCs. To discriminate between the two, we performed ISH against Olfm4 mRNA, another standard and highly robust marker of CBC cells 4. Mex3a-deleted crypts also exhibited a very low amount of Olfm4 transcripts (Fig 2I), thus reinforcing the evidence for Lgr5+ ISCs loss. We also assessed the mRNA expression level of the +4 cell markers Lrig1 8 and Hopx 10 and found a significantly reduced expression of the former in Mex3a-deleted crypts (Fig 2J and K), indicating that there is no compensatory mechanism enforced by the reserve stem cell population in this context. These results were further validated by quantitative real-time PCR (qPCR) expression analysis of the different stem cell markers using isolated crypt fractions from Mex3a KO and WT mice (Fig 2L). In order to directly examine the effect of Mex3a deletion on the Lgr5+ I
Referência(s)