Deletion of the RNaseIII Enzyme Dicer in Thyroid Follicular Cells Causes Hypothyroidism with Signs of Neoplastic Alterations

Micro-RNAs (miRNAs) are small non-coding RNAs that regulate gene expression, mainly at mRNA post-transcriptional level. Functional maturation of most miRNAs requires processing of the primary transcript by Dicer, an RNaseIII-type enzyme. To date, the importance of miRNA function for normal organogenesis has been demonstrated in several mouse models of tissue-specific Dicer inactivation. However, the role of miRNAs in thyroid development has not yet been addressed. For the present study, we generated mouse models in which Dicer expression has been inactivated at two different stages of thyroid development in thyroid follicular cells. Regardless of the time of Dicer invalidation, the early stages of thyroid organogenesis, preceding folliculogenesis, were unaffected by the loss of small RNAs, with a bilobate gland in place. Nevertheless, Dicer mutant mice were severely hypothyroid and died soon after weaning unless they were substituted with T4. A conspicuous follicular disorganization was observed in Dicer mutant thyroids together with a strong down regulation of Nis expression. With increasing age, the thyroid tissue showed characteristics of neoplastic alterations as suggested by a marked proliferation of follicular cells and an ongoing de-differentiation in the center of the thyroid gland, with a loss of Pax8, FoxE1, Nis and Tpo expression. Together, our data show that loss of miRNA maturation due to Dicer inactivation severely disturbs functional thyroid differentiation. This suggests that miRNAs are mandatory to fine-tune the expression of thyroid specific genes and to maintain thyroid tissue homeostasis

Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1

BACKGROUND: The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood. RESULTS: By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis. CONCLUSION: In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Paracrine communications control reorganization of epithelial masses into polarized monolayers

The pancreas, submandibular and thyroid glands originally derive from the endoderm and undergo two opposite epithelial transitions to reach their mature tissue organization. During the first transition, endodermal cells at specified sites loose polarity and proliferate so as to generate an undifferentiated three-dimensional cell mass. In the second transition, the bud reorganizes to form polarized epithelial monolayers. A final choice of epithelial architecture is lumen fusion into branched tubes (exocrine pancreas and submandibular glands) vs closed follicles (endocrine thyroid). The aim of my thesis was to better understand the cellular and molecular mechanisms controlling the second epithelial transition. I first described precisely this transition in the three organs, and found that epithelial cells composing the mass are always in close contact with the mesenchyme and the endothelium during reorganization into polarized monolayers. This raised the possibility of paracrine communications between these different cell types. In the pancreas and submandibular glands, we found that the chemokine Stromal cell-Derived Factor-1 (SDF-1) is produced by the mesenchyme while its main receptor is expressed by the epithelium. SDF-1 binding to CXCR4 is necessary for branching morphogenesis of both organs. Of further interest, SDF-1 production by the submandibular mesenchyme could also target the CXCR7-expressing blood vessels, which, in turn, produce an as yet unidentified signal that promotes submandibular gland branching. Combining my results on thyroid development with those on pancreas development reveals that VEGF-A expression by the epithelium may be either regionalized (restricted to the trunk cells in the pancreas) or global (all epithelial cells of the thyroid). Accordingly, VEGFR2+ endothelial cells are either recruited only near trunk cells in the pancreas, or uniformly in the thyroid. Using transgenic mouse models and an original thyroid explant culture system, we showed that endothelial cells signal back to the epithelium. In the pancreas, they restrict acinar differentiation to tip cells and support endocrine progenitors. In the thyroid, endothelial cells promote folliculogenesis and expression of calcitonin by C-cells. We further demonstrated in the thyroid that these effects depend on a secreted factor released by endothelial cells, rather than on contacts. In conclusion, we have shown that reciprocal paracrine interactions govern the reorganization of a mass of epithelial cells into polarized monolayers. In particular: (i) mesenchymal SDF-1 controls pancreas and submandibular glands branching; (ii) epithelial VEGFA recruits blood vessels; and (iii) blood vessels control pancreas differentiation and thyroid gland morphogenesis and differentiation.(SBIM 3) -- UCL, 201

An Ex vivo Culture System to Study Thyroid Development.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the "angio-follicular" units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood. This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo. Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR. In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis

Serum Levels of Coll2-1, a Specific Biomarker of Cartilage Degradation, Are Not Affected by Sampling Conditions, Circadian Rhythm, and Seasonality.

OBJECTIVE: To assess intraindividual biological variability of serum cartilage specific biomarker Coll2-1 and define the best standardized conditions for blood sampling. DESIGN: Blood samples were taken from 116 subjects with knee osteoarthritis (OA) at a single time point (PRODIGE study) and from 15 healthy subjects under various conditions, including fasting condition, sampling time and season, blood treatment, and type of blood collection tube (COVAR study). Type II collagen-specific biomarker Coll2-1 was directly measured in serum using an immunoassay. RESULTS: There was no significant difference on Coll2-1 values between samples collected at any of the 5 sampling times or at any of the sampling days measured. None of the sampling parameters tested had a significant impact on Coll2-1 value (clotting time, clotting temperature and temperature of blood centrifugation, type of tube). On the contrary, differences were found in between subjects and between subjects with knee OA and healthy subjects. CONCLUSION: Coll2-1 measurement is not affected by sampling specific conditions, circadian rhythm or seasons but was found elevated in subject with knee OA indicating that Coll2-1 serum variation is not linked to the study environment, but to cartilage degradation in OA. Coll2-1 assay is sufficiently robust for use in OA clinical trials

An oleuropein-based dietary supplement improves joint functionality in older people with high knee joint pain

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Epithelial : endothelial cross-talk regulates exocrine differentiation in developing pancreas

Endothelial cells are required to initiate pancreas development from the endoderm. They also control the function of endocrine islets after birth. Here we investigate in developing pancreas how the endothelial cells become organized during branching morphogenesis and how their development affects pancreatic cell differentiation. We show that endothelial cells closely surround the epithelial bud at the onset of pancreas morphogenesis. During branching morphogenesis, the endothelial cells become preferentially located near the central (trunk) epithelial cells and remain at a distance from the branch tips where acinar cells differentiate. This correlates with predominant expression of the angiogenic factor Vascular Endothelial Growth Factor-A (VEGF-A) in trunk cells. In vivo ablation of VEGF-A expression by pancreas-specific inactivation of floxed Vegfa alleles results in reduced endothelial development and in excessive acinar differentiation. On the contrary, acinar differentiation is repressed when endothelial cells are recruited around tip cells that overexpress VEGF-A. Treatment of embryonic day 12.5 explants with VEGF-A or with VEGF receptor antagonists confirms that acinar development is tightly controlled by endothelial cells. We also provide evidence that endothelial cells repress the expression of Ptf1a, a transcription factor essential for acinar differentiation, and stimulate the expression of Hey-1 and Hey-2, two repressors of Ptf1a activity. In explants, we provide evidence that VEGF-A signaling is required, but not sufficient, to induce endocrine differentiation. In conclusion, our data suggest that, in developing pancreas, epithelial production of VEGF-A determines the spatial organization of endothelial cells which, in turn, limit acinar differentiation of the epithelium

Early conditional Dicer inactivation in the developing thyroid causes hypothyroidism.

<p><b>A</b>: To verify that Dicer is inactivated in the thyroid, a PCR was performed in genomic DNA isolated from thyroid from 4 weeks old mice. The different genotypes correspond to: wt (+/+) <i>Dicer^flox/flox</i>, <i>Pax8(Cre/+)</i>; <i>Dicer^flox/flox</i> (−/−) and <i>Pax8(Cre/+)</i>; <i>Dicer^flox/+</i> (+/−). <i>Dicer</i> excision upon <i>cre</i> recombination resulted in an absence of a visible fragment that was observed in homozygous mice, whereas there is a single 1,300 bp fragment in the wt and the heterozygous mice. <b>B</b>: Growth delay in <i>Pax8(Cre/+)</i>; <i>Dicer^flox/flox</i> (−/−) age matched animals. <b>C</b>: Low concentration of total blood T4 in <i>Pax8(Cre/+)</i>; <i>Dicer^flox/flox</i> mice (p = 0,001, evaluated by a t test). <b>D</b>: Elevated TSH in <i>Pax8(Cre/+)</i>; <i>Dicer^flox/flox</i> mice. <b>E, F</b>: Loss of follicular structure in thyroid sections of <i>Pax8(Cre/+)</i>; <i>Dicer^flox/flox</i> (−/−) 3 weeks old mice.</p