Role of Notch signalling in intestinal epithelium

Résumé: Chez les mammifères, les intestins sont les organes ayant le plus haut taux de renouvellement cellulaire dans l'organisme. L'épithélium intestinal se renouvelle complètement en moins d'une semaine. Il se compose de projections (villosités) et d'invaginations (cryptes) qui ont toutes deux des fonctions bien distinctes. Les cellules de l'intestin sont constamment produites à partir de cellules souches, situées dans la crypte, qui se différencient en cellules proliférantes transitoires, puis en cellules caliciformes, de Paneth, entéroendocrine ou en entérocytes. Ces cellules migrent dans leurs lieux spécifiques pour accomplir leur fonction physiologique pour finalement mourir. A cours de mon travail de thèse, j'ai étudié le rôle de la voie de signalisation de Notch dans le renouvellement cellulaire et dans le processus de l'homéostase des cellules de l'intestin marin en utilisant le système Cre-loxP pour induire la délétion des gènes Notch1, Notch2, Jaggedl et RBP-Jk. Bien que l'inactivation de Notch1 avec ou sans Jagged1, ou celle de Notch2, n'aboutissent à aucun phénotype, une déficience pour RBP-Jk, ou pour Notch1 et Notch2 simultanément, conduit au développement d'un impressionnant phénotype. Au niveau de la crypte, une rapide et importante modification des cellules apparaît: les cellules proliférantes sont devenues des cellules caliciformes qui ont perdu la capacité de se renouveler. Ces résultats impliquent la voie Notch en tant que nouvelle clé de voûte dans le maintien des cellules qui s'auto-renouvellent dans l'épithélium intestinal. Un rôle similaire a été proposé pour la voie Wnt, laquelle n'est cependant, pas affectée dans nos souris. C'est pourquoi ces deux voies sont essentielles dans le maintien de la prolifération dans les cryptes intestinales. Ce travail a aussi proposé un mécanisme par lequel la voie Notch contrôlerait l'intégrité du cycle cellulaire dans les cellules de la crypte intestinale, ceci en inhibant la transcription d'un inhibiteur du cycle cellulaire, la protéine p27KIP1. De plus, l'inactivation de RBP-Jk dans les adénomes développés par les souris APCmin induisent la différenciation de cellules tumorales en cellules caliciformes. Comme autre effet, la localisation histologique des cellules de Paneth est également affectée par la délétion de RBP-Jk ou de Notch1/Notch2, suggérant un rôle pour la voie Notch dans le compartiment des cellules de Paneth. Finalement, ce travail démontre que les cellules progénitrices de l'intestin ont besoin d'une convergence fonctionnelle des voie Wnt et Notch. Ces résultats préliminaires peuvent être considérés comme un concept pour l'utilisation d'inhibiteurs de secrétase-γ (inhibiteurs de Notch) à des fins thérapeutiques pour les cancers colorectaux. Summary The mammalian intestine has one of the highest cellular turnover rates in the body. The complete intestinal epithelium is renewed in less than a week. It is divided into spatially distinct compartments in the form of finger-like projections (villi) and flask-shaped invaginations (crypts) that are dedicated to specific functions. Intestinal cells are constantly produced from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate and home to their specific compartments before dying after having fulfilled their physiological function. In this thesis project, the physiological role of the Notch signalling cascade in the marine intestine was studied. Inducible tissue specific inactivation of Notch1, Notch2, Jagged1 and RBP-Jk genes was applied to assess their role in the maintenance of intestinal homeostasis and cell fate determination. The analysis unequivocally revealed that Notch1, Notch1 and Jagged1 combined as well as Notch2 are dispensable for intestinal homeostasis and lineage differentiation. However, deficiency of RBP-Jk as well as the simultaneous inactivation of both Notch1 and Notch2 receptors unveiled a striking phenotype. In these mice, a rapid and massive conversion of proliferative crypt cells into post-mitotic goblet cells was observed. These results identify the Notch pathway as a key player for the maintenance of the proliferative crypt compartment. A similar role was implicated for the Wnt cascade, which, however, was not affected in the different tissue specific Notch signalling deficient mice. Thus, the Wnt and Notch signalling pathways are essential for the self-renewal capacity of the intestinal epithelium. Furthermore, our results suggest a molecular mechanism for Notch signalling mediated control of cell cycle regulation within the crypt. The Notch cascade inhibits expression of the cyclin-dependent kinase inhibitor p27KIP1 and thereby maintains proliferation of the intestinal progenitor cells. In addition, the inactivation of RBP-Jk in adenomas developed by APCmin mice resulted in the differentiation of tumour cells into goblet cells. Finally, Notch deficiency affected differentiated Paneth cells, suggesting that Notch may play a role in the Paneth cell compartment. In summary, this work clearly demonstrates that undifferentiated, proliferative cells in intestinal crypts require the concerted activation of the RBP-Jk-mediated Notch signalling and the Wnt cascade. In addition, our preliminary results can be considered as a "proof-of-principle" for the use of γ-secretase inhibitors for therapeutic modalities for colorectal cancer.

Indocyanine Green Angiography Findings in Acute Retinal Necrosis Syndrome.

BACKGROUND: Acute retinal necrosis syndrome is clinically defined by the presence of peripheral necrotizing retinitis associated with severe occlusive vasculitis caused primarily by herpes simplex virus and varicella zoster virus. Previously considered as an exclusively retinal pathology, choroidal involvement, as demonstrated by indocyanine green angiography, has not been extensively studied. HISTORY AND SIGNS: Indocyanine green angiography was performed in 4 patients with ARN. Observed angiographic patterns included: 1. a characteristic triangular area of hypo-perfusion, 2. hypofluorescent lobular patches and areas of fuzzy choroidal vascular hyperfluorescence, and 3. isolated hypofluorescent lobular patches of the contralateral eye. THERAPY AND OUTCOME: Marked choroidal hypo-perfusion on indocyanine green angiography was associated with extensive retinal ischemia. Treatment included a combination of antiviral agents and corticosteroids complemented by prophylactic acetylsalicylate. CONCLUSION: Indocyanine green angiography may provide important information regarding choroidal vascular involvement in ARN. It may also permit the timely identification of sub-clinical contralateral eye involvement.

Chromosomal number aberrations and transformation in adult mouse retinal stem cells in vitro.

PURPOSE: The potential of stem cells (SCs) as a source for cell-based therapy on a wide range of degenerative diseases and damaged tissues such as retinal degeneration has been recognized. Generation of a high number of retinal stem cells (RSCs) in vitro would thus be beneficial for transplantation in the retina. However, as cells in prolonged cultivation may be unstable and thus have a risk of transformation, it is important to assess the stability of these cells. METHODS: Chromosomal aberrations were analyzed in mouse RSC lines isolated from adult and from postnatal day (PN)1 mouse retinas. Moreover, selected cell lines were tested for anchorage-dependent proliferation, and SCs were transplanted into immunocompromised mice to assess the possibility of transformation. RESULTS: Marked aneuploidy occurred in all adult cell lines, albeit to different degrees, and neonatal RSCs were the most stable and displayed a normal karyotype until at least passage 9. Of interest, the level of aneuploidy of adult RSCs did not necessarily correlate with cell transformation. Only the adult RSC lines passaged for longer periods and with a higher dilution ratio underwent transformation. Furthermore, we identified several cell cycle proteins that might support the continuous proliferation and transformation of the cells. CONCLUSIONS: Adult RSCs rapidly accumulated severe chromosomal aberrations during cultivation, which led to cell transformation in some cell lines. The culture condition plays an important role in supporting the selection and growth of transformed cells.

A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

Cells derived from the coelomic epithelium contribute to multiple gastrointestinal tissues in mouse embryos.

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1(cre)) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms' tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1(cre-YFP) cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1(cre-YFP) cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1(cre-YFP) mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.

Reproducibility of straylight measurement by C-Quant for assessment of retinal straylight using the compensation comparison method.

BACKGROUND: Straylight gives the appearance of a veil of light thrown over a person's retinal image when there is a strong light source present. We examined the reproducibility of the measurements by C-Quant, and assessed its correlation to characteristics of the eye and subjects' age. PARTICIPANTS AND METHODS: Five repeated straylight measurements were taken using the dominant eye of 45 healthy subjects (age 21-59) with a BCVA of 20/20: 14 emmetropic, 16 myopic, eight hyperopic and seven with astigmatism. We assessed the extent of reproducibility of straylight measures using the intraclass correlation coefficient. RESULTS: The mean straylight value of all measurements was 1.01 (SD 0.23, median 0.97, interquartile range 0.85-1.1). Per 10 years of age, straylight increased in average by 0.10 (95%CI 0.04 to 0.16, p < 0.01]. We found no independent association of refraction (range -5.25 dpt to +2 dpt) on straylight values (0.001; 95%CI -0.022 to 0.024, p = 0.92). Compared to emmetropic subjects, myopia reduced straylight (-.011; -0.024 to 0.02, p = 0.11), whereas higher straylight values (0.09; -0.01 to 0.20, p = 0.09) were observed in subjects with blue irises as compared to dark-colored irises when correcting for age. The intraclass correlation coefficient (ICC) of repeated measurements was 0.83 (95%CI 0.76 to 0.90). CONCLUSIONS: Our study showed that straylight measurements with the C-Quant had a high reproducibility, i.e. a lack of large intra-observer variability, making it appropriate to be applied in long-term follow-up studies assessing the long-term effect of surgical procedures on the quality of vision.

Intrinsically photosensitive retinal ganglion cell function in relation to age: a pupillometric study in humans with special reference to the age-related optic properties of the lens.

BACKGROUND: The activity of melanopsin containing intrinsically photosensitive ganglion retinal cells (ipRGC) can be assessed by a means of pupil responses to bright blue (appr.480 nm) light. Due to age related factors in the eye, particularly, structural changes of the lens, less light reaches retina. The aim of this study was to examine how age and in vivo measured lens transmission of blue light might affect pupil light responses, in particular, mediated by the ipRGC. METHODS: Consensual pupil responses were explored in 44 healthy subjects aged between 26 and 68 years. A pupil response was recorded to a continuous 20 s light stimulus of 660 nm (red) or 470 nm (blue) both at 300 cd/m2 intensity (14.9 and 14.8 log photons/cm2/s, respectively). Additional recordings were performed using four 470 nm stimulus intensities of 3, 30, 100 and 300 cd/m2. The baseline pupil size was measured in darkness and results were adjusted for the baseline pupil and gender. The main outcome parameters were maximal and sustained pupil contraction amplitudes and the postillumination response assessed as area under the curve (AUC) over two time-windows: early (0-10 s after light termination) and late (10-30 s after light termination). Lens transmission was measured with an ocular fluorometer. RESULTS: The sustained pupil contraction and the early poststimulus AUC correlated positively with age (p=0.02, p=0.0014, respectively) for the blue light stimulus condition only.The maximal pupil contraction amplitude did not correlate to age either for bright blue or red light stimulus conditions.Lens transmission decreased linearly with age (p<0.0001). The pupil response was stable or increased with decreasing transmission, though only significantly for the early poststimulus AUC to 300 cd/m2 light (p=0.02). CONCLUSIONS: Age did not reduce, but rather enhance pupil responses mediated by ipRGC. The age related decrease of blue light transmission led to similar results, however, the effect of age was greater on these pupil responses than that of the lens transmission. Thus there must be other age related factors such as lens scatter and/or adaptive processes influencing the ipRGC mediated pupil response enhancement observed with advancing age.

Intrinsically photosensitive retinal ganglion cells.

The recent discovery of melanopsin-expressing retinal ganglion cells that mediate the pupil light reflex has provided new insights into how the pupil responds to different properties of light. These ganglion cells are unique in their ability to transduce light into electrical energy. There are parallels between the electrophysiologic behavior of these cells in primates and the clinical pupil response to chromatic stimuli. Under photopic conditions, a red light stimulus produces a pupil constriction mediated predominantly by cone input via trans-synaptic activation of melanopsin-expressing retinal ganglion cells, whereas a blue light stimulus at high intensity produces a steady-state pupil constriction mediated primarily by direct intrinsic photoactivation of the melanopsin-expressing ganglion cells. Preliminary data in humans suggest that under photopic conditions, cones primarily drive the transient phase of the pupil light reflex, whereas intrinsic activation of the melanopsin-expressing ganglion cells contributes heavily to sustained pupil constriction. The use of chromatic light stimuli to elicit transient and sustained pupil light reflexes may become a clinical pupil test that allows differentiation between disorders affecting photoreceptors and those affecting retinal ganglion cells.

IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies.

The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified in ABCA4, CNGB1, GUCY2D, PROM1, PRPF8, PRPF31, PRPH2, RHO, RP2, and TULP1 for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies.

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy.

OBJECTIVE: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). MATERIALS AND METHODS: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. RESULTS: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. CONCLUSION: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

The rationale of retinal endovascular fibrinolysis in the treatment of retinal vein occlusion : from experimental data to clinical application.

PURPOSE: : We describe a retinal endovascular fibrinolysis technique to directly reperfuse experimentally occluded retinal veins using a simple micropipette. METHODS: : Retinal vein occlusion was photochemically induced in 12 eyes of 12 minipigs: after intravenous injection of 10% fluorescein (1-mL bolus), the targeted retinal vein segment was exposed to thrombin (50 units) and to Argon laser (100-200 mW) through a pars plana approach. A beveled micropipette with a 30-μm-diameter sharp edge was used for micropuncture of the occluded vein and endovascular microinjection of tissue plasminogen activator (50 μg/mL) in 11 eyes. In one control eye, balanced salt solution was injected. The lesion site was examined histologically. RESULTS: : Retinal vein occlusion was achieved in all cases. Endovascular microinjection of tissue plasminogen activator or balanced salt solution led to reperfusion of the occluded retinal vein in all cases. Indicative of successful reperfusion were the following: continuous endovascular flow, unaffected collateral circulation, no optic disk ischemia, and no venous wall bleeding. However, balanced salt solution injection was accompanied by thrombus formation at the punctured site, whereas no thrombus was observed with tissue plasminogen activator injection. CONCLUSION: : Retinal endovascular fibrinolysis constitutes an efficient method of micropuncture and reperfusion of an experimentally occluded retinal vein. Thrombus formation at the punctured site can be prevented by injection of tissue plasminogen activator.

Pigment production by Streptococcus agalactiae in quasi-defined media.

A quasi-defined medium that supports the growth of Streptococcus agalactiae as pigmented colonies has been developed. The medium contains starch, a peptic digest of albumin, amino acids, nucleosides, vitamins, and salts. The presence of free cysteine, which could be replaced with other sulphur-containing compounds and to a lesser degree by reducing agents, was required for pigment formation.