Direct communication of neighboring cells by gap junction channels is essential for the development of tissues and organs in the body. Whereas vertebrate gap junctions are composed of members of the connexin family of transmembrane proteins, in invertebrates gap junctions consist of Innexin channel proteins. Innexins display very low sequence homology to connexins. In addition, very little is known about their cellular role during developmental processes. In this report, we examined the function and the distribution of Drosophila Innexin 2 protein in embryonic epithelia. Both loss-of-function and gain-of-function innexin 2 mutants display severe developmental defects due to cell death and a failure of proper epithelial morphogenesis. Furthermore, immunohistochemical analyses using antibodies against the Innexins 1 and 2 indicate that the distribution of Innexin gap junction proteins to specific membrane domains is regulated by tissue specific factors. Finally, biochemical interaction studies together with genetic loss- and gain-of-function experiments provide evidence that Innexin 2 interacts with core proteins of adherens and septate junctions. This is the first study, to our knowledge, of cellular distribution and protein-protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila.

Ceramide synthases are highly conserved transmembrane proteins involved in the biosynthesis of sphingolipids, which are essential structural components of eukaryotic membranes and can act as second messengers regulating tissue homeostasis. However, the role of these enzymes in development is poorly understood due to the lack of animal models. We identified schlank as a new Drosophila member of the ceramide synthase family. We demonstrate that schlank is involved in the de novo synthesis of a broad range of ceramides, the key metabolites of sphingolipid biosynthesis. Unexpectedly, schlank mutants also show reduction of storage fat, which is deposited as triacylglyerols in the fat body. We found that schlank can positively regulate fatty acid synthesis by promoting the expression of sterol-responsive element-binding protein (SREBP) and SREBP-target genes. It further prevents lipolysis by downregulating the expression of triacylglycerol lipase. Our results identify schlank as a new regulator of the balance between lipogenesis and lipolysis in Drosophila. Furthermore, our studies of schlank and the mammalian Lass2 family member suggest a novel role for ceramide synthases in regulating body fat metabolism.

Maintenance of metabolic homeostasis requires adaption of gene regulation to the cellular energy state via transcriptional regulators. Here, we identify a role of ceramide synthase (CerS) Schlank, a multiple transmembrane protein containing a catalytic lag1p motif and a homeodomain, which is poorly studied in CerSs, as a transcriptional regulator. ChIP experiments show that it binds promoter regions of lipases lipase3 and magro via its homeodomain. Mutation of nuclear localization site 2 (NLS2) within the homeodomain leads to loss of DNA binding and deregulated gene expression, and NLS2 mutants can no longer adjust the transcriptional response to changing lipid levels. This mechanism is conserved in mammalian CerS2 and emphasizes the importance of the CerS protein rather than ceramide synthesis. This study demonstrates a double role of CerS Schlank as an enzyme and a transcriptional regulator, sensing lipid levels and transducing the information to the level of gene expression.

Edited by Laszlo NagyDrosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-b import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.Keywords: ceramide synthase; Drosophila; homeodomain; importin; lipid homeostasis; nuclear function Ceramides and sphingolipids are key intermediates in diverse cellular processes for growth, stress resistance, apoptosis, neurodegeneration, insulin function, and lipid homeostasis [1][2][3][4][5].In synthesis of ceramide, Ceramide Synthases (CerSs) acylate a sphingoid long-chain base (LCB) with a fatty acyl-CoA of distinct length to generate (dihydro) ceramide, the backbone of all complex sphAbbreviations aa, amino acid; ASAH1, acid ceramidase; ATF2, activating transcription factor 2; cDNA, complementary DNA; CerSs, ceramide synthases; dsRNA, double-stranded RNA; ER, endoplasmic reticulum; Gal4, galactose-responsive transcription factor; GFP, green fluorescent protein; GlcT-1, glucosylceramide synthase; H215D, change in a histidine at position 215 of schlank into glutamate; INM, inner nuclear membrane;

Endoreduplication cycles that lead to an increase of DNA ploidy and cell size occur in distinct spatial and temporal patterns during Drosophila development. Only little is known about the regulation of these modified cell cycles. We have investigated fore- and hindgut development and we present evidence that the Drosophila knirps and knirps-related genes are key components to spatially restrict endoreduplication domains. Our lack and gain-of-function experiments show that knirps and knirps-related which encode nuclear orphan receptors transcriptionally repress S-phase genes of the cell cycle required for DNA replication and that this down-regulation is crucial for gut morphogenesis. Furthermore, we demonstrate that both genes are activated in overlapping expression domains in the fore- and hindgut in response to Wingless and Hedgehog activities emanating from epithelial signaling centers that control the regionalization of the gut tube. Our results provide a novel link between morphogen-dependent positional information and the spatio-temporal regulation of cell cycle activity in the gut.