Enterocytes Research Articles

Nutrient excess in a stabilized co-culture system of Caco2/HT-29 cells: an ultrastructural and functional time-course study

The intestine represents one of the most important barriers of our body and interacts with several exogenous substances, among which nutrients. Today, the effects due to an excess of nutrients on intestinal morpho-functional changes, similar to the ones found in obesity, have been studied only in in vivo animal models. Many experimental difficulties hampered in establishing a physiological long-term experimental model starting from primary cultures of normal small intestinal and colon cells. For this reason, an intestinal Caco2/HT-29 (70/30) co-culture was set up in our lab starting from the differentiated parental cell populations to mimic the human intestinal epithelium. Co-culture was harvested at confluence (T0) and at 3, 7, and 15 days (T3, T7, and T15, respectively) post-confluence. Ultrastructural (TEM) and functional analysis (Alkaline Phosphatase, ALP; Aminopeptidase N, APN; Dipeptidyl Peptidase-IV , DPPIV; Transepithelial Electrical Resistance, TEER) were carried out. In the present study, two parallel experimental groups were cultured: the standard group and the excess group. In the standard group, the culture medium was changed every four days, whilst in the excess group on alternate days from T0. Transmission electron microscopy revealed that the excess of nutrients drives co-cultures towards a less differentiated absorptive phenotype. On the other hand, mucus granule presence was more and more evident from T3. The specific activity of ALP and APN, known markers of intestinal differentiation, and that of DPPIV, a specific marker of enterocyte differentiation, progressively increased. TEER, indicative of the barrier properties of the co-culture, increased at post confluence up to T15. In conclusion, data here presented show that the excess of nutrients can directly modify both morphology and function of the intestinal cells, opening the way to study at the effects due to specific nutrients on cell proliferation and differentiation involved in the acquisition of an obese human phenotype.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.

Intestinal Stem Cell Pool Regulation in Drosophila.

SummaryIntestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, we performed partial ISC depletion. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. We discovered, however, that the loss of differentiated midgut enterocytes (ECs) slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. Our study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis.

The Life and Death of Differentiated and Dedifferentiated Intestinal Absorptive and Secretory Cells

Intestinal stem cells (ISC) differentiate into many types of secretory and absorptive progenies which migrate up the villi and are exfoliated after 3–5 d. Because epithelial homeostasis requires balanced rates of proliferation, migration, differentiation and apoptosis, studies increasing our understanding of cell turnover are critical. We used murine primary intestinal organoids to determine lifespan of different intestinal cell types. We tested the hypotheses that organoids directed to differentiate into distinct cell types would exhibit lifespans similar to those previously estimated in vivo, and that forced dedifferentiation of organoids containing mature cells would result in reacquisition of ISC characteristics and extension of lifespan. We directed, via Wnt and Notch signaling, strict ISC differentiation into specialized organoids comprised primarily of stem cells (ISC), enterocytes (ENT), goblet cells (GOB), or Paneth cells (PAN). Cell type enrichment was confirmed via biomarker mRNA expression and immunofluorescence, as follows: stem cells (~95%), enterocytes (~90%), goblet (~85%) or Paneth cells (~65%) which contain relative proportions much higher than in situ.We found that continuously proliferating ISC organoids can be passaged several times; in contrast, ENT and GOB organoids do not live past 5 or 4 d, respectively, and PAN organoids live ~11 d. These findings are similar to known in vivo lifespans. During differentiation, ENT and GOB organoids exhibited a gradual increase in expression of enterocyte biomarker sucrase isomaltase and goblet cell biomarker mucin‐2, respectively, while levels of stem cell markers Leucine Rich Repeat Containing G Protein‐Coupled Receptor 5 (Lgr5) and SRY‐Box 9 (Sox9) decreased dramatically. Forced dedifferentiation of ENT and GOB organoids was characterized by a dramatic decrease in expression of differentiated cell markers paralleled by pronounced increases in stem cell characteristics.Differentiation was accompanied by sharp decreases over time in anti‐apoptotic B‐cell lymphoma 2 (Bcl2) in both ENT and GOB organoids (>90%). Changes in expression of pro‐apoptotic caspase‐3 (Casp3) and Fas Cell Surface Death Receptor (Fas) were insignificant. Dedifferentiated ENT organoids (dENT) expressed altered apoptotic markers compared to their normal ENT and ISC counterparts. Casp3 and Fas levels decreased 50 and 70% respectively in dENT organoids compared to ENT controls while Bcl2 and Tumor protein p53 (P53) increased significantly (1.6 and 5‐fold respectively). In dedifferentiated goblet organoids (dGOB), Bcl2 and P53 also increased 2–3‐fold. Phenotypically, life‐span increased markedly in both dENT and dGOB organoids by more than 2‐fold due to dedifferentiation, but cell morphology was not altered.In conclusion, we were able to differentiate organoids enriched in distinct cell lineages, then to induce these specialized organoids to undergo in vitro dedifferentiation which markedly extended their life span. In vitro lifespan of specific cell type organoids was similar to that in vivo, and was strongly correlated with mRNA levels of Bcl2. Additionally, we were able to induce specialized organoids to undergo in vitro dedifferentiation and markedly extend their lifespan. This study furthers our understanding of organoid physiology, intestinal cell turnover, and plasticity of cell fate.Support or Funding InformationSupported by NSF Grants No. IOS‐1121049 and 1456673 (RPF). RPF also received support from NIH R01‐DK‐102934 (NG).

Intestinal stem cell ablation reveals differential requirements for survival in response to chemical challenge

The Drosophila intestine is maintained by multipotent intestinal stem cells (ISCs). Although increased intestinal stem cell (ISC) proliferation has been correlated with a decrease in longevity, there is some discrepancy regarding whether a decrease or block in proliferation also has negative consequences. Here we identify headcase (hdc) as a novel marker of ISCs and enteroblasts (EBs) and demonstrate that Hdc function is required to prevent ISC/EB loss through apoptosis. Hdc depletion was used as a strategy to ablate ISCs and EBs in order to test the ability of flies to survive without ISC function. While flies lacking ISCs showed no major decrease in survival under unchallenged conditions, flies depleted of ISCs and EBs exhibited decreased survival rates in response to damage to mature enterocytes (EC) that line the intestinal lumen. Our findings indicate that constant renewal of the intestinal epithelium is not absolutely necessary under normal laboratory conditions, but it is important in the context of widespread chemical-induced damage when significant repair is necessary.

Probiotic Bacteria and their Supernatants Protect Enterocyte Cell Lines from Enteroinvasive Escherichia coli (EIEC) Invasion.

Probiotic microorganisms have attracted a growing interest for prevention and therapy of gastrointestinal disorders. Many probiotic strains have been shown to inhibit growth and metabolic activity of enteropathogenic bacteria as well as their adhesion and invasion to intestinal cells. In the present study, we evaluated the interference of bacteria-free supernatants (BFS) of cultures belonging to sixteen strains of lactobacilli and bifidobacteria, with invasion of enteroinvasive Escherichia coli (EIEC) strain, using human colonic adenocarcinoma cell lines, T84 and Caco2 cells. To assess invasion of Caco-2 and T84 cells by EIEC, and measure the number of pathogens inside the enterocytes, the gentamicin protection assay was conducted. In addition, three different invasion inhibition assays were designed; namely co-incubation, pre-incubation and treatment with the BFS of probiotics. Data obtained and theoretical calculation showed that the most effective assay in the prevention of pathogen invasion was treatment with BFS. Besides, co-incubation assay was more valid than pre-incubation assay in invasion prevention. The obtained results suggest that probiotics may produce some metabolites that strongly prevent invasion of enteroinvasive E.coli into the small and large intestine. Also, probiotics are able to compete with or exclude pathogen invasion.

MiR-263a Regulates ENaC to Maintain Osmotic and Intestinal Stem Cell Homeostasis in Drosophila

Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinalstem cell (ISC) homeostasis. We found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Finally, we provide evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.

Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis.

Ageing results in loss of tissue homeostasis across taxa. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinaldiseases.

Intestinal IRE1 Is Required for Increased Triglyceride Metabolism and Longer Lifespan under Dietary Restriction

Dietary restriction (DR) is one of the most robust lifespan-extending interventions in animals. The beneficial effects of DR involve a metabolic adaptation toward increased triglyceride usage. The regulatory mechanism and the tissue specificity of this metabolic switch remain unclear. Here, we show that theIRE1/XBP1 endoplasmic reticulum (ER) stress signaling module mediates metabolic adaptation upon DR in flies by promoting triglyceride synthesis and accumulation in enterocytes (ECs) of the Drosophila midgut. Consistently, IRE1/XBP1 function in ECs is required for increased longevity upon DR. We further identify sugarbabe, a Gli-like zinc-finger transcription factor, as a key mediator of the IRE1/XBP1-regulated induction of de novo lipogenesis in ECs. Overexpression of sugarbabe rescues metabolic and lifespan phenotypes of IRE1 loss-of-function conditions. Our study highlights the critical role of metabolic adaptation of the intestinal epithelium for DR-induced lifespan extension and explores the IRE1/XBP1 signaling pathway regulating this adaptation and influencing lifespan.

تأثیر تراکم مواد مغذی و اسید آمینۀ ال- گلوتامین افزودنی بر عملکرد رشد، مورفولوژی روده کوچک و پاسخ ایمنی جوجههای گوشتی

این مطالعه به منظور بررسی اثر سطوح مختلف تراکم مواد مغذی و گلوتامین افزودنی بر عملکرد رشد، مورفولوژی ژژنوم و پاسخ‌های ایمنی جوجه‌های گوشتی در دورۀ آغازین (10-0 روزگی)، رشد (24-11 روزگی) و پایانی (42-25 روزگی) انجام گرفت. آزمایشی با 320 قطعه جوجه گوشتی نر سویه راس 308 در قالب یک طرح کاملا تصادفی با 8 تیمار، 4 تکرار و 10 قطعه جوجه در هر تکرار انجام شد. در این آزمایش از دو سطح تراکم مواد مغذی (توصیه راس و 5 درصد رقیق شده) و 4 سطح گلوتامین (0، 5/0، 1 و 5/1 درصد) در قالب طرح کاملا تصادفی به صورت فاکتوریل 2×4 استفاده شد. نتایج آزمایش نشان داد که در دورۀ رشد و پایانی، تراکم مواد مغذی توصیه شده و گلوتامین افزودنی در جیرۀ غذایی موجب بهبود ضریب تبدیل خوراک می‌شود. افزودن گلوتامین موجب افزایش وزن نسبی ژژنوم، کل روده کوچک، تیموس و بورس گردید. سطح تراکم مواد مغذی و گلوتامین بر ارتفاع ویلی و عمق کریپت ژژنوم تأثیر معنی‌داری داشت. در بررسی ایمنی هومورال، بیشترین تیتر آنتی‌بادی بر علیه SRBC اولیه و ثانویه در تیمار حاوی 5/1 درصد گلوتامین و کمترین آن در گروه شاهد (بدون گلوتامین افزودنی) مشاهده شد. با تزریق زیر جلدی فیتوهماگلوتینین- P مشخص شد که گلوتامین افزودنی موجب افزایش ضخامت پردۀ بین انگشتان پای پرندگان گردید. بطور کلی نتایج حاصل از این تحقیق نشان داد که با تنظیم جیره‌های ‌غذایی با تراکم مواد مغذی توصیه شده برای سویه راس و افزودن 5/0 درصد گلوتامین، عملکرد رشد بهبود می‌یابد و تغذیه جوجه‌ها با جیرۀ حاوی 1 درصد گلوتامین افزودنی و تراکم مواد مغذی توصیه شده پاسخ‌های ایمنی ارتقاء می‌یابد.

PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis

Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy.

Co-infection with EHEC and C. albicans in vitro enhances invasion and tissue damage of enterocytes induced by C. albicans

Objective To investigate, in vitro, the co-infection of Caco-2 cells (epithelial cells of intestinal mucosa) with Candida albicans and Enterohemorrhagic escherichia coli (EHEC). Methods The ability of both species to invade the Caco-2 cells was evaluated by inverted microscopy. Damage to Caco-2 cells was evaluated by measuring lactate dehydrogenase (LDH) activity. C. albicans virulence gene expression (ALS3, PLB1 and SAP4) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Results Compared to simple infections with C.albicans alone, a co-infection invaded Caco-2 cells more rapidly, and C. albicans tended to proliferate more easily presenting in cluster shape of distribution. In addition, the LDH activity in the co-infection group (group 3) was the highest compared to groups 1, 2, 4 and 5, (F values of 14.48, 5.48, 11.74 and 3.45 respectively; all P <0.05); There was no significant difference in LDH activity found between the secondary fungous infection group (group 5) and the EHEC infection group (group 2) (F=2.03, P=0.54) or between the secondary bacterial infection group (group 4) and the Candida albicans infection group (group 1) (F=2.74, P=0.11). The LDH activities in groups 2 and 5 were significantly higher than that in groups 1 and 4 (all P <0.05). In addition, an up-regulation of toxicity-related genes (PLB1 and SAP4) were detected. The expression of PLB1 was higher in group 3 than that in group 1(P=0.014 3)and SAP4 was higher in groups 3 and 5 than that in group 1(P=0.027 2, P=0.001 8, respectively). Conclusions Using Caco-2 cells for an infection model, this study demonstrated that co-infecting in vitro enterocytes with C.albicans and EHEC enhanced the invasiveness and tissue damaging effects of C.albicans. Key words: Enterohemorrhage escherichia coli; Candida albicans; Co-infection; Invasion; Tissue damage; Inverted microscope; LDH; qRT-PCR

GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult midgut

Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation.

Hindsight/RREB-1 functions in both the specification and differentiation of stem cells in the adult midgut of Drosophila.

ABSTRACTThe adult Drosophila midgut is established during the larval/pupal transition from undifferentiated cells known as adult midgut precursors (AMPs). Four fundamental cell types are found in the adult midgut epithelium: undifferentiated intestinal stem cells (ISCs) and their committed daughter cells, enteroblasts (EBs), plus enterocytes (ECs) and enteroendocrine cells (EEs). Using the Drosophila posterior midgut as a model, we have studied the function of the transcription factor Hindsight (Hnt)/RREB-1 and its relationship to the Notch and Egfr signaling pathways. We show that hnt is required for EC differentiation in the context of ISC-to-EC differentiation, but not in the context of AMP-to-EC differentiation. In addition, we show that hnt is required for the establishment of viable or functional ISCs. Overall, our studies introduce hnt as a key factor in the regulation of both the developing and the mature adult midgut. We suggest that the nature of these contextual differences can be explained through the interaction of hnt with multiple signaling pathways.

Notch role in multipotency or cell fate

Stem Cell Regulation Multipotent Drosophila intestinal stem cells (ISCs) generate either nutrient-absorbing enterocytes (ECs) or secretory enteroendocrine cells (EECs). Guo and Ohlstein investigated the role of Notch signaling in this process. They tracked ISC asymmetric divisions and found that EEC daughter cells, which have a low level of Notch, signal back to the ISC in order to keep it multipotent. However, during EC production, ISCs activate strong Notch signaling in daughters. Thus, Notch signaling functions in two directions to achieve stem cell multipotency. Science , this issue p. [10.1126/science.aab0988][1] [1]: /lookup/doi/10.1126/science.aab0988

Stem cell regulation. Bidirectional Notch signaling regulates Drosophila intestinal stem cell multipotency.

Drosophila intestinal stem cells (ISCs) generate enterocytes (ECs) and enteroendocrine (ee) cells. Previous work suggests that different levels of the Notch ligand Delta (Dl) in ISCs unidirectionally activate Notch in daughters to control multipotency. However, the mechanisms driving different outcomes remain unknown. We found that during ee cell formation, the ee cell marker Prospero localizes to the basal side of dividing ISCs. After asymmetric division, the ee daughter cell acts as a source of Dl that induces low Notch activity in the ISC to maintain identity. Alternatively, ISCs expressing Dl induce high Notch activity in daughter cells to promote EC formation. Our data reveal a conserved role for Notch in Drosophila and mammalian ISC maintenance and suggest that bidirectional Notch signaling may regulate multipotency in other systems.

Abstract 1705: Classification of colorectal PDX into transcriptomic subtypes associated with distinct genomic alteration profiles and in vivo response patterns to therapies

Abstract Recently, unsupervised gene expression-based signatures with prognostic and potential predictive implications were proposed for colorectal cancer (CRC) classification. The challenge is now to characterize the genomic alterations of CRC subtypes and their sensitivity to therapies. Patient derived xenograft models (PDX) may be a valuable tool for this purpose, as they retain the molecular features and drug response patterns of their parental patient tumors. In this study, we classified our collection of colon PDX into transcriptomic subtypes and investigated the associations with genomic alterations and in vivo responses to cetuximab (CTX), oxaliplatin (OXT) and irinotecan (IR). We determined expression profiles of 67 PDX models using Affymetrix HGU133 Plus2.0 arrays and applied the CRC assigner-786 gene expression signature reported by Sadanandam et al. to classify our PDX into 5 subtypes. We identified 34 (51%) transit-amplifying (TA), 15 (22%) goblet-like (GL), 10 (15%) inflammatory (IF), 7 (10%) enterocyte (ET) and 1 (1%) stem-like (SL) PDX models. TA showed a gene signature of WNT pathway activation whereas GL, ET and IF PDX subtypes harbored gene signatures of KRAS pathway activation. As analyzed by whole exome sequencing and Affymetrix SNP6.0 array, the GL, ET and IF subtypes were found to display different signatures of mutational processes, mutations and chromosomal rearrangement patterns than TA and SL. Of particular note, mutations in BRAF, PIK3CA/PTEN, TGFBR2/SMAD4 or NOTCH1 were mainly found in GL, ET or IF while mutations in APC, TP53 and KRAS were not associated with a given subtype. Regarding PDX drug sensitivity, 10/11 TA/SL PDX with unaltered KRAS/PIK3CA/PTEN/HER2 status were sensitive to CTX whereas 9/12 models with alterations were resistant. TA and SL PDX were also frequently sensitive to IR and OXT (6/10 and 4/10, respectively). In the IF subtype, 6/6 PDX showing KRAS, PIK3CA or PTEN alterations were resistant to CTX. Of interest, 5/5 IF PDX were sensitive to IR treatment. As in IF PDX, GL and ET often had alterations in KRAS/PIK3CA/PTEN/HER2 and were resistant to CTX. Moreover, these subtypes were resistant to both IR and OXT. Furthermore, the frequent KRAS pathway activation and the presence of actionable mutations in GL, ET and IF would advocate the testing of inhibitors (as monotherapy or in combination) of BRAF, PI3K or MEK in tumors of these subtypes while WNT inhibitors should be tested in TA models.Molecular classification of our colon PDX collection confirmed their similarities with patient tumors. Investigation of drug sensitivity resulted in the identification of PDX subtypes which respond poorly to standard therapies and which require different treatment options. Testing alternative and combined therapies in PDX may be a valuable approach to optimize personalized medicine. Citation Format: Anne-Lise Peille, Christina Gredy, Bruno Zeitouni, Armin Maier, Kerstin Klingner, Tim Kees, Julia Schüler, Thomas Metz, Heinz. Herbert Fiebig, Vincent Vuaroqueaux. Classification of colorectal PDX into transcriptomic subtypes associated with distinct genomic alteration profiles and in vivo response patterns to therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1705. doi:10.1158/1538-7445.AM2015-1705

Regional Cell-Specific Transcriptome Mapping Reveals Regulatory Complexity in the Adult Drosophila Midgut

Deciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1-R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, we generated RNA-sequencing transcriptomes of all five cell types isolated by FACS from each of the five regions, R1-R5. In doing so, we identify transcriptional diversities among cell types and document regional differences within each cell type that define further specialization. We validate cell-specific and regional Gal4 drivers; demonstrate roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation, and study the transcriptional response of midgut cells upon infection. The resulting transcriptome database (http://flygutseq.buchonlab.com) will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions.

Gut-neuron interaction via Hh signaling regulates intestinal progenitor cell differentiation in Drosophila.

Intestinal homeostasis is maintained by intestinal stem cells (ISCs) and their progenies. A complex autonomic nervous system spreads over posterior intestine. However, whether and how neurons regulate posterior intestinal homeostasis is largely unknown. Here we report that neurons regulate Drosophila posterior intestinal homeostasis. Specifically, downregulation of neuronal Hedgehog (Hh) signaling inhibits the differentiation of ISCs toward enterocytes (ECs), whereas upregulated neuronal Hh signaling promotes such process. We demonstrate that, among multiple sources of Hh ligand, those secreted by ECs induces similar phenotypes as does neuronal Hh. In addition, intestinal JAK/STAT signaling responds to activated neuronal Hh signaling, suggesting that JAK/STAT signaling acts downstream of neuronal Hh signaling in intestine. Collectively, our results indicate that neuronal Hh signaling is essential for the determination of ISC fate.

The changes of gene expression of iron transporters for duodenal iron uptake and export in diet-induced obese mice

This study aims to determine the gene expression changes of iron transporters-divalent metal transporter 1 (DMT1) and ferroportin 1 (Fpn1) in the duodenal tissue of diet-induced obese mice. C57BL/6J mice were randomly divided into normal control (NC) and obesity model (OM) group, 6 in each, and fed on conventional and high-fat diet respectively for 14 weeks by table of random number. Then the DMT1 and Fpn1 mRNA contents in duodenal tissues of the animals were measured by Real-time PCR method, and the protein expression levels were analyzed by Western blot test. The Real-time PCR detection results showed that, compared with the NC group for which the mRNA expression level was defined as 1.0, the Fpn1 mRNA expression in OM group (0.58±0.11) was reduced significantly (t = 6.71, P = 0.014), whereas the relative expression level of DMT1 mRNA in OM group (0.89±0.26) showed no obvious alteration (t = 2.01, P = 0.122). Western blot results showed that the relative protein expression levels of Fpn1 in OM and NC group were 0.32±0.06 and 0.65±0.19, respectively, and the difference was statistically significant (t = 5.37, P = 0.026). The DMT1 protein relative abundance was 0.88±0.21 in OM group and 0.92±0.17 in NC group, and the difference has no statistical significance (t = 1.84, P = 0.185). Fpn1 gene expression is inhibited in the duodenum of diet-induced obesity mouse while DMT1 expression keeps unchanged, and this implies that decreased iron export from enterocytes into circulation might be responsible for the impaired iron absorption in obesity.