The ability to establish spatial organization is an essential feature of any developing tissue and is achieved through well-defined rules of cell-cell communication. Maintenance of this organization requires elimination of cells with inappropriate positional identity, a poorly understood phenomenon. Here we studied mechanisms regulating cell elimination in the context of a growing tissue, the Drosophila wing disc and its dorsal determinant Apterous. Systematic analysis of apterous mutant clones along with their twin spots shows that they are eliminated from the dorsal compartment via three different mechanisms: relocation to the ventral compartment, basal extrusion, and death, depending on the position of the clone in the wing disc. We find that basal extrusion is the main elimination mechanism in the hinge, whereas apoptosis dominates in the pouch and in the notum. In the absence of apoptosis, extrusion takes over to ensure clearance in all regions. Notably, clones in the hinge grow larger than those in the pouch, emphasizing spatial differences. Mechanistically, we find that limiting cell division within the clones does not prevent their extrusion. Indeed, even clones of one or two cells can be extruded basally, demonstrating that the clone size is not the main determinant of the elimination mechanism to be used. Overall, we revealed three elimination mechanisms and their spatial biases for preserving pattern in a growing organ.

Generation of polyclonal antibodies specifi c to the ribosomal protein S6 kinase isoform-p85S6K1 and directed to the N-terminal (1-23 aa) extension of p85S6K1. Methods. Animal immunization with synthetic (1-23 aa) peptide, ELISA, western blot, immunoprecipitation, immunofl uorescent analysis. Results. Polyclonal antibodies have been generated, which specifi cally recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofl uorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension-the identifi cation of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms. K e y w o r d s: Ribosomal protein S6 kinase 1 (S6K1), S6K1 isoforms, p70 S6K1, p85 S6K1, polyclonal antibodies.

Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1-64aa and 14-64aa Nterminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies.

15The ability to establish spatial organization is an essential feature of any developing tissue and 16 is achieved through well-defined rules of cell-cell communication. Maintenance of this 17 organization requires elimination of cells with inappropriate positional identity, a poorly 18 understood phenomenon. Here we studied mechanisms regulating cell elimination in the 19 context of a growing tissue, the Drosophila wing disc and its dorsal determinant Apterous. 20 Systematic analysis of apterous mutant clones along with their twin spots shows that they are 21 eliminated from the dorsal compartment via three different mechanisms: sorting to the 22 ventral compartment, basal extrusion, and death, depending on the position of the clone in 23 the wing disc. We find that basal extrusion is the main elimination mechanism in the hinge, 24 whereas apoptosis dominates in the pouch and in the notum. In the absence of apoptosis, 25 extrusion takes over to ensure clearance in all regions. Notably, clones in the hinge grow larger 26 than those in the pouch, emphasizing spatial differences. Mechanistically, we find that limiting 27 cell division within the clones does not prevent their extrusion. Indeed, even clones of one or 28 two cells can be extruded basally, demonstrating that the clone size is not the main 29 determinant of the elimination mechanism to be used. Overall, we revealed three elimination 30 mechanisms and their spatial biases for preserving pattern in a growing organ. 31 33 34Multicellularity requires precise spatial organization of cells during development. 35 Aberrant cells that arise as a result of sporadic mutations or chromatin defects can challenge 36