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Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis
© Sibon et al; licensee BioMed Central Ltd. 2008
Received: 05 June 2008
Accepted: 29 August 2008
Published: 29 August 2008
Coenzyme A (CoA) is an essential metabolite, synthesized from vitamin B5 by the subsequent action of five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK. Mutations in Drosophila dPPCS disrupt female fecundity and in this study we analyzed the female sterile phenotype of dPPCS mutants in detail.
We demonstrate that dPPCS is required for various processes that occur during oogenesis including chorion patterning. Our analysis demonstrates that a mutation in dPPCS disrupts the organization of the somatic and germ line cells, affects F-actin organization and results in abnormal PtdIns(4,5)P2 localization. Improper cell organization coincides with aberrant localization of the membrane molecules Gurken (Grk) and Notch, whose activities are required for specification of the follicle cells that pattern the eggshell. Mutations in dPPCS also induce alterations in scutellar patterning and cause wing vein abnormalities. Interestingly, mutations in dPANK and dPPAT-DPCK result in similar patterning defects.
Together, our results demonstrate that de novo CoA biosynthesis is required for proper tissue morphogenesis.
Coenzyme A (CoA), the major acyl carrier in all organisms, constitutes an essential cofactor to support cellular metabolism . Synthesis of CoA occurs via a conserved route in which vitamin B5 is subsequently modified by five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK [2–5]. Although CoA biosynthesis is well characterized in bacteria and in in vitro systems , only recently has the impact of abnormal CoA biosynthesis on animals been investigated [7–10].
Mutations in dPPCS impair female fecundity and fertility
Previously, we isolated a Drosophila dPPCS mutant as a female sterile, neurologically impaired mutant and we demonstrated that CoA metabolism is required to maintain DNA integrity during development of the central nervous system . Here, we analyzed the female sterile phenotype of a hypomorphic allele of dPPCS (dPPCS1) in detail. dPPCS33 mutants (a null allele) are homozygous lethal , and in dPPCS1/33 mutants, no vitellogenic egg chambers were observed. Using immunohistochemistry and confocal laser scanning microscopy (supplement) we have analyzed the defects that occur during oogenesis (see for recent reviews [11, 12]).
Between 144–192 h AE, dPPCS1/1 females deposited 0.03 (± 0.02 SEM) eggs/24 h, none of which hatched (n = 142 eggs), while wt females produced 10.0 (± 1.4 SEM) eggs/24 h, of which 90% hatched (n = 1005 eggs). It has been reported that a mid-oogenesis checkpoint monitors the integrity of pre-vitellogenic egg chambers, and that activation of this checkpoint results in the removal of abnormal egg chambers . A Tunnel assay was performed, which revealed that in dPPCS1/1 ovaries at 144 h AE, prior to vitellogenesis, a 6-fold increase of ovariols containing apoptotic egg chambers was observed, compared to wt ovaries (see additional file 1). Approximately 32% of dPPCS1/1 ovariols (n = 222) contained stage 5–7 egg chambers that displayed packaging defects (abnormal amount of germ line cells), while 4% of the wt ovariols (n = 109) contained egg chambers with packaging defects. When we expressed a dPPCS transgene (P[dPPCS]) in the dPPCS1/1 background, 11% (n = 166) of the ovariols displayed defects, demonstrating that dPPCS is required for early egg chamber development. Within dPPCS1/1 germaria, aberrant separation of the developing egg chambers by the intercyst cells likely results in production of egg chambers with abnormal interfollicular stalk cell and/or polar follicle cell formation, egg chambers with mispositioned oocytes, or egg chambers that display packaging defects (see additional file 1). Thus, the reduced fecundity of the dPPCS1/1 females is most likely due to production of aberrant egg chambers that did not pass the mid-oogenesis checkpoint and were absorbed.
dPPCS is required for F-actin remodeling during cytoplasmic dumping
Mutations in dPPCS affect egg chamber development, stage 10–11 F-actin organization & cytoplasmic dumping
% of egg chambers
Nurse cells trapped inside the oocyte
0.0 (n = 100)
20.9 (n = 67)
1.8 (n = 56)
F-actin clumps in ooplasm
3.0 (n = 100)
50.1 (n = 53)
7.0 (n = 57)
Aberrant F-actin in nurse cells
0.0 (n = 100)
92.2 (n = 51)
29.5 (n = 61)
Nurse cells plugging ring canals
0.0 (n = 100)
71.0 (n = 62)
15.5 (n = 58)
Oocytes with disorganized subcortical F-actin
0.0 (n = 100)
43.7 (n = 55)
0.0 (n = 53)
Oocyte nuclei with F-actin fibers
0.0 (n = 100)
18.8 (n = 48)
0.0 (n = 46)
Grk and Notch localization is disrupted in dPPCS 1/1 egg chambers
We hypothesized that disorganized tissue integrity may also affect the signaling routes required for specification of follicle cells that pattern the chorion. To investigate this, we stained ovaries with antibodies against Notch and Grk, which both are required for specification of the follicle cell populations that pattern the eggshell [14, 21]. Although we cannot conclude that Grk or Notch signaling was disrupted in dPPCS1/1 ovaries, the localization of both proteins was frequently impaired compared to wt ovaries. In wt egg chambers, when the border cells reach the centripetal follicle cells, Notch is highly expressed at the dorsoanterior corner, where it is required for the specification of the dorsal appendage producing cells, while Notch expression is restricted to the nurse cell membranes during cytoplasmic dumping (Fig. 4Ba, see additional file 1) . In dPPCS1/1 stage 11 egg chambers, Notch localization was more diffuse throughout the nurse cells and not restricted to the membranes (Fig. 4Ca). Notch localization was also severely affected during late stage oogenesis (see additional file 1) and FasIII staining revealed that the dorsal appendage/operculum forming follicle cells were not properly organized (see additional file 1).
In wt stage 9–10 egg chambers, Grk is localized at the dorsoanterior corner of the oocyte compartment. Although in dPPCS1/1 egg chambers, Grk was present at the dorsoanterior corner, the distribution of the protein was frequently impaired in stage 8–9 egg chambers (see additional file 1) and progressively worsened when egg chambers proceeded into later stages of oogenesis (see additional file 1).
These findings imply that dPPCS is not required for cell specification or signaling per se, but merely required for cell organization and morphology. This is supported by the finding that aberrant intercyst cell migration/organization likely underlies the observed packaging and follicle cell specification defects during early oogenesis (see additional file 1).
Membrane localization of PtdIns(4,5)P2 is impaired in dPPCS1/1
The levels of phospholipids are reduced in dPPCS mutant flies, indicating a general defect in phospholipid biosynthesis . Therefore, it is plausible to assume that phosphatidylinositol (PtdIns) production, the precursor for all phosphoinositides , is also reduced. Although levels and localization of PtdIns have not been determined during Drosophila oogenesis, it is generally accepted that actin remodeling processes depend on PtdIns signaling .
Although the F-actin/PtdIns(4,5)P2 connection should be investigated in more detail, we propose that F-actin remodeling within the Drosophila ovary likely depends on PtdIns(4,5)P2 signaling and that this lipid derived signaling route is disrupted in dPPCS1/1. Abnormal cytoskeletal organization in dPPCS1/1 disrupts the overall shape of all membranous structures and the organization of the cells during morphogenesis. Disorganized tissue integrity could affect Notch and Grk localization and possibly signaling, which is required for specification of the follicle cells that pattern the eggshell, and causes severe chorion patterning defects.
dPPCS is required for patterning of various tissues
Next, we wondered whether dPPCS is also required for morphogenesis of other tissues. Hereto, we closely investigated dPPCS1/1 flies for other morphological abnormalities. A stereotypical pattern of four scutellars exists on the dorsal surface of the wt scutellum, and dPPCS mutants displayed ectopic formation of scutellars (see additional file 1). Furthermore, dPPCS1/1 flies also developed ectopic wing veins (see additional file 1). Mutants initiated longitudinal vein formation between L3–L4 and L4–L5. These results show that dPPCS is required for morphogenesis of various tissues during Drosophila development.
Mutations in de novo CoA synthesis disrupt morphogenesis
Next, we investigated whether mutations in other CoA biosynthesis enzymes give rise to similar defects. Indeed, mutations in dPANK/fumble and the bifunctional enzyme dPPAT-DPCK result in similar characteristics compared to the dPPCS mutant phenotype. dPANK/fumble and dPPAT-DPCK mutant females have poorly developed ovaries, have fecundity defects, produce eggs that exhibit polarity defects, synthesize abnormal neutral lipids (droplets), and these mutants display scutellar and wing vein patterning defects (see additional file 1). As in dPPCS1/1, a mutation in dPPAT-DPCK disrupts actin localization and results in plugging of the ring canals by nurse cell nuclei during dumping (see additional file 1). dPANK/fumble mutants produce small ball-shaped eggs, which are typically due to a loss of actin regulatory elements that control the polarized arrangement of F-actin fibers at the basal cortex of follicle cells required to establish planar cell polarity . These findings imply that impaired CoA synthesis in general disrupts morphogenesis, possibly due to aberrant F-actin organization. Because the biosynthesis route towards the production of CoA is conserved amongst species it would be interesting to explore the significance of CoA during processes that involve actin/PtdIns dynamics such as chemotaxis, axon growth cone guidance, endocytosis/exocytosis, cell division or actin dependent chromatin remodeling.
We thank L. Cooley and A. Wodarz for the UAS-PLCδ-PH-GFP line and S. Wasserman for the P[dPANK] line. This work was supported by a VIDI grant from the Netherlands Organization for Scientific Research (NWO; 971-36-400) to O.C.M.S and by a Topmaster grant from the Graduate School GUIDE to A.R.
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