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Ectodermal organs, which include teeth, hair follicles, mammary ducts, and glands such as sweat,mucous and sebaceous glands, are initiated in development as placodes, which are epithelial thickenings that invaginate and bud into the underlying mesenchyme. These placodes are stratified into a basal and several suprabasal layers of cells. The mechanisms driving stratification and invagination are poorly understood. Using the mouse molar tooth as a model for ectodermal organ morphogenesis, we show here that vertical, stratifying cell divisions are enriched in the forming placode and that stratification is cell division dependent. Using inhibitor and gain-of-function experiments, we show that FGF signalling is necessary and sufficient for stratification but not invagination as such.We show that, instead, Shh signalling is necessary for, and promotes, invagination once suprabasal tissue is generated. Shh-dependent suprabasal cell shape suggests convergent migration and intercalation, potentially accounting for post-stratification placode invagination to bud stage. We present a model in which FGF generates suprabasal tissue by asymmetric cell division, while Shh triggers cell rearrangement in this tissue to drive invagination all the way to bud formation. [ABSTRACT FROM AUTHOR]

Salivary glands are branching organs which develop by bud and cleft formation to create an organ with a large surface area. The epithelium and mesenchyme signal back and forth to control this branching process, with additional cues provided by the parasympathetic nerves and blood vessels that surround the developing branches. This branching morphogenesis can be recapitulated successfully in organ culture , allowing access to the tissue to follow development and manipulate the tissue interactions, and signals. To culture glands, the filter-grid method has been widely used, allowing the development of salivary glands cultured as a whole organ, or the gland epithelium in isolation, or with the surrounding craniofacial tissue in a cranial slice. Here, we describe the methods for each approach and show the applicability of culturing glands from a wide variety of species: mouse , snake, and human. The resulting samples and data from these cultures can be employed for morphological and molecular analysis, with some examples described in this chapter, bringing valuable knowledge to our understanding of branching morphogenesis., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Salivary glands are formed by branching morphogenesis with epithelial progenitors forming a network of ducts and acini (secretory cells). During this process, epithelial progenitors specialise into distal (tips of the gland) and proximal (the stalk region) identities that produce the acini and higher order ducts, respectively. Little is known about the factors that regulate progenitor expansion and specialisation in the different parts of the gland. Here, we show that Sox9 is involved in establishing the identity of the distal compartment before the initiation of branching morphogenesis. Sox9 is expressed throughout the gland at the initiation stage before becoming restricted to the distal epithelium from the bud stage and throughout branching morphogenesis. Deletion of Sox9 in the epithelium results in loss of the distal epithelial progenitors, a reduction in proliferation and a subsequent failure in branching. We demonstrate that Sox9 is positively regulated by mesenchymal Fgf10 , a process that requires active Erk signalling. These results provide new insights into the factors required for the expansion of salivary gland epithelial progenitors, which can be useful for organ regeneration therapy., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)