The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner

Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner. Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. We analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. Here we show that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. We also show that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf

Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex

LIM-Homeodomain (LIM-HD) genes encode a family of transcription factors known to be involved in development and patterning in several systems. Previously, we have shown that LIM-HD gene Lhx2 is required for the formation of a crucial boundary in the dorsal telencephalon (Bulchand et al. [2001] Mech Dev 100:165-175). To further explore the role of LIM-HD genes as well as the broader LIM gene family in dorsal telencephalic development, we examined the expression pattern of the members of this gene family and their cofactors in the developing mouse cerebral cortex. Transcription factor activity of the LIM-HD proteins requires the formation of a tetrameric complex consisting of two LIM-HD molecules linked by a dimer of cofactor (Clim) molecules. LIM-only (Lmo) proteins can interfere with this process by competing for the cofactors. LIM-HD protein function, thus, can be modulated by the presence of the appropriate Clim or Lmo molecules. At least 13 LIM-HD, 4 Lmo, and 2 Clim genes have been identified in the mouse. Several of these genes exhibit complex spatiotemporal patterns spanning different stages of cortical development, from embryonic to postnatal ages. Noteworthy features of the expression patterns include delineation of boundaries within the developing cortex, up- or down-regulation during formation of selected cortical layers, and a striking complementarity of expression of several members consistent with specific functions in cortical development. Significantly, in some cases, Lmo or Clim gene expression is robust where no LIM-HD gene expression is detectable. These results suggest multiple and distinct roles for LIM-HD, Lmo, and Clim genes in cortical development, and also support a LIM-HD-independent role for some Lmo and Clim members

The <it>recombination activation gene 1 </it>(<it>Rag1</it>) is expressed in a subset of zebrafish olfactory neurons but is not essential for axon targeting or amino acid detection

Abstract Background Rag1 (Recombination activation gene-1) mediates genomic rearrangement and is essential for adaptive immunity in vertebrates. This gene is also expressed in the olfactory epithelium, but its function there is unknown. Results Using a transgenic zebrafish line and immunofluorescence, we show that Rag1 is expressed and translated in a subset of olfactory sensory neurons (OSNs). Neurons expressing GFP under the Rag1 promoter project their axons to the lateral region of the olfactory bulb only, and axons with the highest levels of GFP terminate in a single glomerular structure. A subset of GFP-expressing neurons contain Gαo, a marker for microvillous neurons. None of the GFP-positive neurons express Gαolf, Gαq or the olfactory marker protein OMP. Depletion of RAG1, by morpholino-mediated knockdown or mutation, did not affect axon targeting. Calcium imaging indicates that amino acids evoke chemotopically organized glomerular activity patterns in a Rag1 mutant. Conclusion Rag1 expression is restricted to a subpopulation of zebrafish olfactory neurons projecting to the lateral olfactory bulb. RAG1 catalytic activity is not essential for axon targeting, nor is it likely to be required for regulation of odorant receptor expression or the response of OSNs to amino acids.</p

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid–induced ceramide biosynthesis in mice

Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid–induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes