Neves, Notch and Hedgehog in the thymus/parathyroid common primordium: Crosstalk in organ f o r m a t i o n , Developmental Biology, http://dx.doi.org/10. 1016/j.ydbio.2016.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractThe avian thymus and parathyroids (T/PT) common primordium derives from the endoderm of the third and fourth pharyngeal pouches (3/4PP). The molecular mechanisms that govern T/PT development are not fully understood. Here we study the effects of Notch and Hedgehog (Hh) signalling modulation during common primordium development using in vitro, in vivo and in ovo approaches. The impairment of Notch activity reduced Foxn1/thymus-fated and Gcm2/Pth/parathyroid-fated domains in the 3/4PP and further compromised the development of the parathyroid glands. When Hh signalling was abolished, we observed a reduction in the Gata3/Gcm2-and Lfng-expression domains at the median/anterior and median/posterior territories of the pouches, respectively. In contrast, the Foxn1 expression- This study offers novel evidence on the role of Notch signalling in T/PT common primordium development, in an Hh-dependent manner.

The thymus generates central immune tolerance by producing self-restricted and self-tolerant T-cells as a result of interactions between the developing thymocytes and the stromal microenvironment, mainly formed by the thymic epithelial cells. The thymic epithelium derives from the endoderm of the pharyngeal pouches, embryonic structures that rely on environmental cues from the surrounding mesenchyme for its development. Here, we review the most recent advances in our understanding of the molecular mechanisms involved in early thymic organogenesis at stages preceding the expression of the transcription factor Foxn1, the early marker of thymic epithelial cells identity. Foxn1-independent developmental stages, such as the specification of the pharyngeal endoderm, patterning of the pouches, and thymus fate commitment are discussed, with a special focus on epithelial–mesenchymal interactions.

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The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation. Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry). The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.