Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.
The small G protein Ras regulates many cell processes, such as gene expression, proliferation, apoptosis, and cell differentiation. Its mutations are associated with one-third of all cancers. Ras functions are mediated, at least in part, by Ral proteins and their downstream effector the Ral-binding protein 1 (RalBP1). RalBP1 is involved in endocytosis and in regulating the dynamics of the actin cytoskeleton. It also regulates early development since it is required for the completion of gastrulation in Xenopus laevis. RalBP1 has also been reported to be the main transporter of glutathione electrophiles, and it is involved in multidrug resistance. Such a variety of functions could be explained by a differential regulation of RalBP1 localization. In this study, we have detected endogenous RalBP1 in the nucleus of interphasic cells. This nuclear targeting is mediated by nuclear localization sequences that map to the N-terminal third of the protein. Moreover, in X. laevis embryos, a C-terminal coiled-coil sequence mediates RalBP1 retention in the nucleus. We have also observed RalBP1 at the level of the actin cytoskeleton, a localization that depends on interaction of the protein with active Ral. During mitosis RalBP1 also associates with the mitotic spindle and the centrosome, a localization that could be negatively regulated by active Ral. Finally, we demonstrate the presence of post-transcriptional and post-translational isoforms of RalBP1 lacking the Ral-binding domain, which opens new possibilities for the existence of Ral-independent functions.
SUMMARYMorphogenesis of the vertebrate facial skeleton depends upon inductive interactions between cephalic neural crest cells (CNCCs) and cephalic epithelia. The nasal capsule is a CNCC-derived cartilaginous structure comprising a ventral midline bar (mesethmoid) overlaid by a dorsal capsule (ectethmoid). Although Shh signalling from the anterior-most region of the endoderm (EZ-I) patterns the mesethmoid, the cues involved in ectethmoid induction are still undefined. Here, we show that ectethmoid formation depends upon Dlx5 and Dlx6 expression in a restricted ectodermal territory of the anterior neural folds, which we name NF-ZA. In both chick and mouse neurulas, Dlx5 and Dlx6 expression is mostly restricted to NF-ZA. Simultaneous Dlx5 and Dlx6 inactivation in the mouse precludes ectethmoid formation, while the mesethmoid is still present. Consistently, siRNA-mediated downregulation of Dlx5 and Dlx6 in the cephalic region of the early avian neurula specifically prevents ectethmoid formation, whereas other CNCC-derived structures, including the mesethmoid, are not affected. Similarly, NF-ZA surgical removal in chick neurulas averts ectethmoid development, whereas grafting a supernumerary NF-ZA results in an ectopic ectethmoid. Simultaneous ablation or grafting of both NF-ZA and EZ-I result, respectively, in the absence or duplication of both dorsal and ventral nasal capsule components. The present work shows that early ectodermal and endodermal signals instruct different contingents of CNCCs to form the ectethmoid and the mesethmoid, which then assemble to form a complete nasal capsule. phenotype has not been yet described in detail (Beverdam et al., 2002; Depew et al., 2002;Levi et al., 2003;Robledo et al., 2002). Noticeably, nasal defects in Dlx5/6 -/-heads are not shared with either single or compound mutations of other Dlx family members (Depew et al., 2005). A recent study has demonstrated that the olfactory pit plays a late role in controlling the morphogenesis of skeletal elements of the avian nasal capsule (Szabo-Rogers et al., 2009). These data were obtained from embryos in which CNCCs had already migrated into the developing olfactory territory and do not provide information about the initial interactions directing CNCCs towards ectethmoid formation. As the olfactory pit derives from the Dlx5/Dlx6-positive anterior part of the neural fold, we decided to explore the possibility that this territory could be endowed with early inductive capacities. Using mouse mutants and surgical and molecular analyses of chick embryos, we demonstrate that an anterior region of the neural fold ectoderm (NF-ZA) is the territory responsible for ectethmoid induction through a Dlx5/Dlx6-dependent mechanism.We conclude that the complete nasal capsule results from the juxtaposition of two independent structures formed by CNCCs competent to respond to different endodermal or ectodermal cues. MATERIALS AND METHODS Avian and mouse embryosAll animal procedures were approved by national and institutional ethical committees. Fertil...
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