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doi: 10.1242/10.1242/dev.00456

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Sply regulation of sphingolipid signaling molecules is essential for Drosophila development

¹ Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614, USA
² Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, USA

View larger version (38K): [in a new window]   Fig. 1. Drosophila homologs of sphingolipid metabolism. De novo synthesis of sphingolipids (blue). Sphingolipid degradative pathway (red). Phospholipid salvage pathway (green). NGI, no gene identified; NHI, no homolog identified. Palmitoyl-CoA is placed in parentheses to indicate the possibility that Drosophila serine palmitoyltransferase may use a short-chain fatty acyl-CoA substrate.

View larger version (64K): [in a new window]   Fig. 2. Sply encodes the Drosophila sphingosine-1-phosphate lyase. (A) CLUSTALW alignment of Sply. The predicted protein product of Sply is 49% and 43% identical and 68% and 60% similar to human and yeast SPL protein sequences respectively. The broken line indicates the putative transmembrane region. The unbroken line indicates a consensus pyridoxal phosphate-binding motif. (B) Sply gene organization. Open reading frames GH13783 and LP04413 and location of transposon insertion (P{PZ}Sply05091) are indicated. (C) Overexpression of Sply in a Saccharomyces cerevisiae SPL mutant restores sphingosine resistance. The LP04413 and GH13783 cDNAs were cloned into yeast expression vector pYES2 and transformed into a yeast SPL mutant strain (dpl1), as described in the Materials and Methods. The transformed strain (dpl1 + Sply) was compared with wild type (DPL1) and SPL mutant overexpressing endogenous yeast SPL (dpl1 + DPL1) strains in a sphingosine resistance assay. Dilutions of saturated cultures for each strain are indicated above. (D) Expression of Sply in a Saccharomyces cerevisiae SPL mutant restores SPL enzyme activity. Whole cell extracts of Saccharomyces cerevisiae wild type, SPL mutant (dpl1) and SPL mutant overexpressing Sply (dpl1 + Sply) strains were analyzed for SPL activity.

View larger version (61K): [in a new window]   Fig. 3. Sply expression. (A) Sply expression is developmentally regulated. Sply mRNA was quantified by RNase protection, as described in the Materials and Methods. Relative expression was determined using ImageQuant software and standardized to the intensity of a ribosomal protein subunit (RpL32) transcript. (B) In situ hybridization of wild-type embryos shows that Sply has strong, transient expression in the syncytial blastoderm (stage 4) that declines to undetectable levels after cellularization. At stage 11-12, Sply mRNA reappears in the midgut/hindgut rudiments where the developing gut is undergoing extensive reorganization. This gut expression persists for the duration of embryogenesis. The absence of any detectable staining in Sply05091 mutants under identical conditions demonstrates probe specificity and reaffirms that there is no Sply expression in this line. (C) Expression of Sply in wild-type (Canton-S), homozygous Sply mutant (Sply05091) and homozygous Sply revertant (Sply14a) lines. RNA was obtained from 0-to 24-hour-old embryos. RpL32 is again used as a loading control. (D) Degradation of endogenous LCBPs. Extracts of wild-type (square) and Sply mutant (diamond) adult flies were analyzed for the ability to degrade Drosophila endogenous phosphorylated long chain bases.

View larger version (125K): [in a new window]   Fig. 4. Thoracic cross-sections. The Canton-S control fly (A) demonstrates the invariant array of six symmetrical pairs of DLMs (arrows). Sply05091 homozygotes often have missing fibers as represented by this sample (B) that has only four fibers in the left hemithorax and two on the right. Excision of the transposon restores the normal complement of DLMs in most cases, but occasional aberrancies were found, as shown in this Sply14a homozygote (C). This thorax has an extra (seventh) fiber on the left side (arrow). Thoraces of lacek05305 homozygotes generally presented with a normal number of DLMs (D), but the morphology was often distorted, owing to the presence of large vacuolar structures that displaced them from their normal positions towards the midline. The addition of either one (E) or two (F) copies of lacek05305 in the Sply05091 homozygous background restored normal musculature in most cases. With a single copy of lacek05305, however, DLMs were generally smaller than wild-type fibers.

View larger version (81K): [in a new window]   Fig. 5. Bisected thoraces viewed with polarized light. Canton-S control flies (A) display the stereotyped configuration of six DLMs (blue). Seventy-nine percent of the hemithoraces from Sply05091 homozygotes have a reduced number of fibers. This representative hemithorax (B) has only three fibers, which exhibit compensatory hypertrophy. Excision of the transposon reduces the occurrence of aberrant fiber count to 13% as shown in this Sply14a homozygote (C). lacek05305 homozygotes generally (76%) have a normal complement of DLMs; however, occasionally only one large fiber is present as shown (D). A single copy of lacek05305 reduces the occurrence of aberrant hemithoraces in the Sply05091 homozygotes to 63% (E), and 92% of Sply05091, lacek05305 double mutants are normal (F). A DLM schematic shown below [adapted, with permission, from Hartenstein (Hartenstein, 1993)].

View larger version (116K): [in a new window]   Fig. 6. Muscles appear to develop normally in Sply05091 homozygous embryos and larvae. Immunofluorescent staining with -Mef2 (A) and -myosin heavy chain (B) suggests that myoblasts differentiate, migrate, and fuse normally to produce a well-patterned array of embryonic muscles. Larval somatic musculature appears normal (C), most notably with respect to the dorsal oblique muscles (white arrows), which serve as templates for the adult dorsal longitudinal muscles. (Note that dorsal acute muscle 1 was ablated to facilitate visualization of DOMs.) Furthermore, transmission electron microscopy (D) reveals that persistent adult DLMs have intact myofibrils (myo) and structurally normal mitochondria (mit) and nuclei (nuc).

View larger version (79K): [in a new window]   Fig. 7. TUNEL stain. Stage 12-15 embryos were assessed for apoptotic cell death. Canton-S control embryos (A) show background staining in the developing gut. Sply05091 homozygotes (B) consistently show an overall increase in TUNEL-positive cells with a notable cluster at the posterior tip (arrow). The frequency of appearance of this cluster was substantially reduced by both excision of the P-element (C) and by introduction of a single copy of lacek05305 (D).

View larger version (99K): [in a new window]   Fig. 8. Genetic rescue of lacek05305 by Sply05091. The lacek05305 allele is almost completely lethal and the few homozygotes that do survive to adulthood have a lifespan of less than 1 week and manifest pronounced morphological defects (A,C). Wings are notched and often fail to inflate, bristles are missing (arrows), and the eyes are often rough with irregular ommatidia. The addition of the Sply05091 allele (B,D) greatly improves viability, and external morphology is indistinguishable from wild-type flies.

View larger version (40K): [in a new window]   Fig. 9. Possible mechanisms of sphingolipid action during Drosophila development. Sphingolipids have a diverse repertoire of cellular effects depending on cell type. This is due to the diversity of downstream effectors, including both intracellular targets and G-protein-coupled receptors, and to the apparently antagonistic actions of different sphingolipids. The ability to rapidly interconvert these lipids provides a convenient `rheostat' to regulate cell fate (reviewed by Pyne and Pyne, 2000). In addition, sphingoid phospholipids are important components of the plasma membrane. Their long, generally saturated fatty acid moieties increase membrane rigidity and are highly concentrated in specialized rafts that serve as structural elements and have been implicated in a number of signal transduction cascades. This figure summarizes only a few of the possible mechanisms by which sphingolipid intermediates may be acting during development.

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