The em esg /em + cells marked by RFP fluorescence were classified as single, doublets and clusters of at least 3 cells and counted using the cell counter Image-J plug-in. Quantification of midgut em esg /em + em pros /em ? cells The em esg /em + cells, marked by RFP fluorescence, were counted in midguts stained with anti-Prospero at 3 days AED and at the third larval stage (5 days AED for control midguts; 7 days AED for silenced midguts). be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction. Introduction Eukaryotic dyskerins are multifunctional proteins that, within the nucleus, associate with three highly conserved proteins (NOP10, NHP2 and GAR1) and one molecule of H/ACA Nipradilol small nucleolar RNA (snoRNA) to form the H/ACA snoRNP machinery required for RNA pseudouridylation1. In this process, dyskerins act as catalytic pseudouridine synthases, directing the isomerization of specific uridines to pseudouridines, while each assembled snoRNA acts as a guide to select the specific site on target RNA2. The most common targets of pseudouridylation are rRNAs, although pseudouridylation can also influence folding and activity of tRNAs, snRNAs and also mRNA3C6. Mammalian dyskerins, through their ability to bind the telomerase RNA component (TERC), participate also Nipradilol to the formation of the active telomerase holoenzyme that is assembled in the Cajal bodies and preserves telomere integrity7. Proper functionality of these ubiquitous proteins is crucial, as testified by the observation that hypomorphic mutations of dyskerin is involved in rRNA processing and pseudouridylation16. Hypomorphic mutations of gene causes developmental delay, defective maturation of rRNA, small body size, alterations of the abdominal cuticle and reduced fertility, implying a key role in growth and developmental processes. Nipradilol In more recent works, tissue-specific silencing was extensively used to reduce the levels of the protein, and resulted in specific alterations of developmental patterns17 and occurrence of localized apoptosis and tissue remodeling18. In order to investigate whether also the key function in Nipradilol stemness homeostasis is evolutively conserved, we drawed our attention to and dyskerin is required for the formation of larval midgut stem cell niches Ubiquitous RNAi-mediated knockdown of Drosophila dyskerin (the MFL protein) causes lethality at the onset of metamorphosis, underlining the crucial role played by this gene on development17, 20. Given that silenced adult flies were not viable, we focussed on larval midguts to check the role specifically played by the protein on the intestinal stem cell lineage. This organ is composed by only three cells types, all maintained by a hierarchically organized stem cell lineage21, 22 and distinguishable on the basis of their morphology and on the differential expression of two key regulatory genes: ((Adult Midgut precursor cells (AMPs). From early larval stages, AMPs increase their number through a series of symmetric divisions and disperse into the midgut epithelium25, 26. Subsequently, at the beginning of the third larval stage, each dispersed AMP undergoes an asymmetric division that generates another AMP cell and a so Rabbit polyclonal to NOD1 called peripheral cell (PC), in which Notch signaling is specifically activated27. The newly formed AMP then undergoes a few rounds of symmetric divisions (from 3 to 5), Nipradilol generating an expanded cluster of AMPs (from 4 to 16 AMPs) closely surrounded by one or two falcet-shaped PCs. This structure, called imaginal island27, represents the transient larval midgut stem niche25, 28, 29. As shown in Fig.?1A, the three midgut distinct cell types (ECs, ees, AMPs clustered into islands) can easily be morphologically distinguished and all express the ubiquitary MFL protein which, as previously described for other tissues16, 30, concentrates in the nucleoli. Upon ubiquitous silencing (genotype: member of the family of transcription factors. In the midgut, expression is limited to components of the islands (AMPs, PCs) and to a subset of ee cells26, 29. Intriguingly, no island was again formed in this.