Marie-Isabelle Garcia

LGR5 deficiency deregulates Wnt signaling and leads to precocious Paneth cell differentiation in the fetal intestine.

The orphan Leucine-rich repeat G protein-coupled receptor 5 (LGR5/GPR49), a target of Wnt signaling, is a marker of adult intestinal stem cells (SC). However, neither its function in the adults, nor during development of the intestine have been addressed yet. In this report, we investigated the role of LGR5 during ileal development by using LGR5 null/LacZ-NeoR knock-in mice. X-gal staining experiments showed that, after villus morphogenesis, Lgr5 expression becomes restricted to dividing cells clustered in the intervillus region and is more pronounced in the distal small intestine. At day E18.5, LGR5 deficiency leads to premature Paneth cell differentiation in the small intestine without detectable effects on differentiation of other cell lineages, nor on epithelial cell proliferation or migration. Quantitative RT-PCR experiments showed that expression from the LGR5 promoter was upregulated in LGR5-null mice, pointing to the existence of an autoregulatory negative feedback loop in intact animals. This deregulation was associated with overexpression of Wnt target genes in the intervillus epithelium. Transcriptional profiling of mutant mice ileums revealed that LGR5 function is associated with expression of SC and SC niche markers. Together, our data identify LGR5 as a negative regulator of the Wnt pathway in the developing intestine.

Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo

Gene inactivation of the orphan G protein‐coupled receptor LGR4, a paralogue of the epithelial‐stem‐cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4‐deficient crypts or progenitors, but not LGR5‐deficient progenitors, die rapidly with marked downregulation of stem‐cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

The afimbrial adhesive sheath encoded by the afa-3 gene cluster of pathogenic Escherichia coli is composed of two adhesins

The afa‐3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea‐associated Escherichia coli strains. It contains six genes (afaA–afaF ), among which the afaE3 gene is known to code for the structural AfaE‐III adhesin (previously designated AFA‐III), whereas no role has yet been identified for the afaD gene product. The afa‐3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE‐III adhesin; however, unlike the AfaE‐III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa‐3 and daa operons. Analyses of the phenotypes conferred by these afa‐3/daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE‐III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE‐III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE‐III, AfaD was found to be a surface‐exposed protein as well as an adhesin; both AfaD and AfaE‐III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.

Characterization of the AfaD-like family of invasins encoded by pathogenic Escherichia coli associated with intestinal and extra-intestinal infections

The afimbrial adhesive sheath, encoded by the afa‐3 gene cluster, is composed of two proteins with different roles in bacterium–HeLa cell interactions. AfaE is required for adhesion and AfaD for internalization. In this study, we found that the AfaD invasin was structurally and functionally conserved among human afa‐expressing strains, independently of AfaE subtype and clinical origin of the Escherichia coli isolate. The AggB protein from enteroaggregative E. coli was also found to be an AfaD‐related invasin. These data suggest that AfaD is the prototype of a family of invasins encoded by adhesion‐associated operons in pathogenic E. coli.

Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.

ABSTRACT Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2+ cells generating gastric spheroids differed from adult glandular Lgr5+ stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5+ stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2+ cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2+ cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program. Summary: Trop2, a marker of gastric fetal glandular epithelium grown ex vivo, is re-expressed upon injury in adult regenerative cells together with a partial fetal-like genetic program.

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the “pro-hair cell” transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

Stem Cells in Adult Homeostasis, Regeneration and Tissue Development of the Digestive Tract Epithelium

The gastrointestinal epithelium is one of the tissues with highest self-renewing rates under steady-state conditions, and it thereby constitutes an excellent model to better understand how tissues maintain homeostasis. In the past decade, intense research in this field has allowed identifying stem cells responsible for this task and has contributed to uncover the main molecular mechanisms associated with self-renewal and differentiation properties of these cells. One of the future challenges is to fully dissect signaling processes associated with adult regeneration to design new therapies in patients with gastro-intestinal disorders. Here, we review recent advances regarding the identity of cells involved in adult homeostasis and regeneration in the small intestine and stomach. The recent characterization of the developmental epithelial progenitors involved in tissue morphogenesis has provided evidences that epithelial remodeling involved in fetal and adult regenerating tissues share common molecular processes.

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea: Lgr4/5 in the developing cochlea

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferationof precursors and promotes the formation of hair cells by up-regulating Atoh1expression. Not much, however, is known about the regulation of Wnt/β-catenin activityin the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated byan interaction between LGR receptors and their ligands from the R-spondin family. Thedeficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality.Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increasesWnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction ofinner and outer hair cells (OHC). Supernumerary hair cells are likely formed due toan up-regulation of the “pro-hair cell” transcription factors Atoh1, Nhlh1, and Pou4f3.Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs inthe heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolongedthe proliferation in the mid-basal turn of E13 cochleae, causing an increase in thenumber of SOX2-positive precursor cells within the pro-sensory domain. The prematuredifferentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficientembryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion.Altogether, our findings suggest that LGR4 and LGR5 play an important role in theregulation of hair cell differentiation in the embryonic cochlea.