Endothelial cells play multiple roles during pancreas organogenesis. First, they are required to instruct endoderm-derived pancreatic progenitor cells to initiate branching morphogenesis. Later, blood vessels promote β-cell differentiation but also limit acinar development. In this work, we show how endothelial cells might signal to pancreatic progenitors and spatially regulate acinar differentiation. Using an ex vivo culture system of undifferentiated E12.5 pancreata, we demonstrate that embryonic endothelial progenitor cells and their conditioned medium prevent the expression of two members of the pro-acinar transcriptional PTF1L-complex. This effect is not mediated by SPARC, a protein abundantly released in the medium conditioned by endothelial progenitors. On the contrary, heterotrimeric laminin-α1β1γ1, also produced by endothelial progenitor cells, can repress acinar differentiation when used on its own on pancreatic explants. Lastly, we found that laminin-α1 is predominantly found in vivo around the pancreatic trunk cells, as compared to the tip cells, at E14.5. In conclusion, we propose that expression or deposition of laminin-α1β1γ1 around the trunk cells, where blood vessels are predominantly localized, prevent acinar differentiation of these cells. On the contrary, transient decreased expression or deposition of laminin-α1β1γ1 around the tip cells would allow PTF1L-complex formation and acinar differentiation.

Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-α1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-α1.

Tight junction complexes are involved in the establishment and maintenance of cell polarity and the regulation of signalling pathways, controlling biological processes such as cell differentiation and cell proliferation. MarvelD3 is a tight junction protein expressed in adult epithelial and endothelial cells. In Xenopus laevis, MarvelD3 morphants present differentiation defects of several ectodermal derivatives. In vitro experiments further revealed that MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behaviour and survival. In this work, we found that MarvelD3 is expressed from early developmental stages in the exocrine and endocrine compartments of the pancreas, as well as in endothelial cells of this organ. We thoroughly characterized MarvelD3 expression pattern in developing pancreas and evaluated its function by genetic ablation. Surprisingly, inactivation of MarvelD3 in mice did not alter development and differentiation of the pancreatic tissue. Moreover, tight junction formation and organization, cell polarization, and activity of the JNK-pathway were not impacted by the deletion of MarvelD3.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

Organogenesis is the phase of embryonic development leading to the formation of fully functional organs. In the case of the thyroid, organogenesis starts from the endoderm and generates a multitude of closely packed independent spherical follicular units surrounded by a dense network of capillaries. Follicular organisation is unique and essential for thyroid function, i.e. thyroid hormone production. Previous in vivo studies showed that, besides their nutritive function, endothelial cells play a central role during thyroid gland morphogenesis. However, the precise mechanisms and biological parameters controlling the transformation of the multi-layered thyroid epithelial primordium into a multitude of single-layered follicles are mostly unknown. Animal studies used to improve understanding of organogenesis are costly and time-consuming, with recognised limitations. Here, we developed and used a 2-D vertex model of thyroid growth, angiogenesis and folliculogenesis, within the open-source Chaste framework. Our in silico model, based on in vivo images, correctly simulates the differential growth and proliferation of central and peripheral epithelial cells, as well as the morphogen-driven migration of endothelial cells, consistently with our experimental data. Our simulations further showed that reduced epithelial cell adhesion was critical to allow endothelial invasion and fission of the multi-layered epithelial mass. Finally, our model also allowed epithelial cell polarisation and follicular lumen formation by endothelial cell abundance and proximity. Our study illustrates how constant discussion between theoretical and experimental approaches can help us to better understand the roles of cellular movement, adhesion and polarisation during thyroid embryonic development. We anticipate that the use of in silico models like the one we describe can push forward the fields of developmental biology and regenerative medicine.

Differential diagnosis of thyroid cancer and benign nodules is still one of the most challenging issues in the field of endocrinology. To overcome overdiagnosis of papillary thyroid carcinomas (PTC) and the consecutive overtreatment of multinodular diseases, the search for easily accessible, sensitive and accurate biomarkers is critical. Several micro-RNAs (miRNAs) freely circulating in peripheral blood or enclosed in extracellular vesicles (EVs) have been proposed as potential biomarkers from non-invasive liquid biopsies. However, protocols are rarely comparable and conflicting data exists in the literature. In this work, we aimed to assess the diagnostic value of 6 micro-RNAs by comparing their expression in thyroid tissue to their abundance in bulk plasma and in plasma-EVs, before and after thyroid surgery. Plasma-EVs were isolated using a sequential density- and size-based fractionation, followed by in-depth characterization, confirming EV purity. Micro-RNA levels were measured by RT-qPCR in thyroid tissue, plasma and plasma-EVs. Among the 6 candidates, only miR-146b-5p and miR-21a-5p displayed a significant differential abundance in purified plasma-derived EVs from patients with PTC and benign disease. However, no difference could be demonstrated in bulk plasma through our cohort of patients. Overall, our work supports the use of a well-defined protocol of plasma-EV miRNAs purification for biomarker discovery, rather than the use of freely-circulating miRNAs in bulk plasma. Our work also demonstrates that standardized pre-analytical and analytical procedures as well as optimized EV-miRNAs detection methods are essential.

Background: The production of thyroid hormones [triiodothyronine (T3), thyroxine (T4)] depends on the organization of the thyroid in follicles, which are lined by a monolayer of thyrocytes with strict apicobasal polarity. This polarization supports vectorial transport of thyroglobulin (Tg) for storage into, and recapture from, the colloid. It also allows selective addressing of channels, transporters, ion pumps, and enzymes to their appropriate basolateral [Na + /Isymporter (NIS), SLC26A7, and Na + /K + -ATPase] or apical membrane domain (anoctamin, SLC26A4, DUOX2, DUOXA2, and thyroperoxidase). How these actors of T3/T4 synthesis reach their final destination remains poorly understood. The PI 3-kinase isoform Vps34/PIK3C3 is now recognized as a main component in the general control of vesicular trafficking and of cell homeostasis through the regulation of endosomal trafficking and autophagy. We recently reported that conditional Vps34 inactivation in proximal tubular cells in the kidney prevents normal addressing of apical membrane proteins and causes abortive macroautophagy. Methods: Vps34 was inactivated using a Pax8-driven Cre recombinase system. The impact of Vps34 inactivation in thyrocytes was analyzed by histological, immunolocalization, and messenger RNA expression profiling. Thyroid hormone synthesis was assayed by 125 I injection and plasma analysis. Results: Vps34 conditional knockout (Vps34 cKO ) mice were born at the expected Mendelian ratio and showed normal growth until postnatal day 14 (P14), then stopped growing and died at *1 month of age. We therefore analyzed thyroid Vps34 cKO at P14. We found that loss of Vps34 in thyrocytes causes (i) disorganization of thyroid parenchyma, with abnormal thyrocyte and follicular shape and reduced PAS + colloidal spaces; (ii) severe noncompensated hypothyroidism with extremely low T4 levels (0.75 -0.62 lg/dL) and huge thyrotropin plasma levels (19,300 -10,500 mU/L); (iii) impaired 125 I organification at comparable uptake and frequent occurrence of follicles with luminal Tg but nondetectable T4-bearing Tg; (iv) intense signal in thyrocytes for the lysosomal membrane marker, LAMP-1, as well as Tg and the autophagy marker, p62, indicating defective lysosomal proteolysis; and (v) presence of macrophages in the colloidal space. Conclusions: We conclude that Vps34 is crucial for thyroid hormonogenesis, at least by controlling epithelial organization, Tg iodination as well as proteolytic T3/T4 excision in lysosomes.

327 words, 350 allowed) 44 45 BACKGROUND: The production of thyroid hormones (T3, T4) depends on thyroid organization 46 in follicles, lined by a monolayer of thyrocytes with strict apico-basal polarity. Polarization 47 supports vectorial transport of thyroglobulin for storage into, and recapture from, the colloid. 48It also allows selective addressing of channels, transporters, pumps and enzymes to their 49 appropriate basolateral (NIS and Na + /K + -ATPase) or apical membrane domain (pendrin, 50 anoctamin, DUOX2, DUOXA2 and TPO). How these actors of T3/T4 synthesis reach their final 51 destination remains poorly understood. Vps34/PIK3C3 is now recognized as a main 52 component in the general control of vesicular trafficking and of cell homeostasis via 53 autophagy. We recently reported that conditional Vps34 inactivation in kidney proximal 54 tubular cells by Pax8-driven excision prevents normal addressing of apical membrane proteins 55 and causes abortive macroautophagy. 56 METHODS:Vps34 was inactivated using a Pax8-driven Cre recombinase system. The impact 57 of Vps34 inactivation in thyrocytes was analyzed by histological, immunolocalization and 58 mRNA expression profiling. Thyroid hormone synthesis was assayed by 125 I injection and by 59 serum plasma analysis. 60 RESULTS: Vps34 cKO mice were born at the expected Mendelian ratio and showed normal 61 growth until postnatal day 14, then stopped growing and died at around 1 month of age. We 62 therefore analyzed thyroid Vps34 cKO before postnatal day 14. We found that loss of Vps34 in 63 thyrocytes causes: (i) disorganization of thyroid parenchyma with abnormal thyrocyte and 64 follicular shape and reduced PAS + colloidal spaces; (ii) impaired 125 I organification at 65 comparable uptake and frequent occurrence of follicles with luminal thyroglobulin but non-66 detectable T4-bearing thyroglobulin; (iii) severe non-compensated hypothyroidism with 67 4 extremely low T4 levels (<0.25 ± 1.5 µg/dL) and huge TSH plasma levels (19,300 ± 10,500 68 mU/L); (iv) intense signal in thyrocytes for the lysosomal membrane marker, LAMP-1, as well 69 as thyroglobulin and the autophagy marker, p62, indicating defective proteolysis. 70 CONCLUSIONS:We conclude that Vps34 is crucial for thyroid hormonogenesis, at least by 71 controlling delivery of apical actors responsible for biogenesis of thyroid hormones on Tg as 72 well as defective proteolytic T3/T4 excision in lysosomes. 73