RESUMO

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.

Assuntos

Glândulas Salivares , Animais , Células Epiteliais , Epitélio , Mesoderma , Camundongos , Morfogênese , Técnicas de Cultura de Órgãos , Glândula Submandibular

RESUMO

During the formation of repetitive ectodermally derived organs such as mammary glands, lateral line and teeth, the tissue primordium iteratively initiates new structures. In the case of successional molar development, new teeth appear sequentially in the posterior region of the jaw from Sox2(+) cells in association with the posterior aspect of a pre-existing tooth. The sequence of molar development is well known, however, the epithelial topography involved in the formation of a new tooth is unclear. Here, we have examined the morphology of the molar dental epithelium and its development at different stages in the mouse in vivo and in molar explants. Using regional lineage tracing we show that within the posterior tail of the first molar the primordium for the second and third molar are organized in a row, with the tail remaining in connection with the surface, where a furrow is observed. The morphology and Sox2 expression of the tail retains characteristics reminiscent of the earlier stages of tooth development, such that position along the A-P axes of the tail correlates with different temporal stages. Sox9, a stem/progenitor cell marker in other organs, is expressed mainly in the suprabasal epithelium complementary with Sox2 expression. This Sox2 and Sox9 expressing molar tail contains actively proliferating cells with mitosis following an apico-basal direction. Snail2, a transcription factor implicated in cell migration, is expressed at high levels in the tip of the molar tail while E-cadherin and laminin are decreased. In conclusion, our studies propose a model in which the epithelium of the molar tail can grow by posterior movement of epithelial cells followed by infolding and stratification involving a population of Sox2(+)/Sox9(+) cells.