Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis

Background 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. Results 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. Conclusion Together, our results demonstrate that de novo CoA biosynthesis is required for proper tissue morphogenesis.

and confocal laser scanning microscopy (supplement) we have analyzed the defects that occur during oogenesis (see for recent reviews [11,12]).
At 48 h after eclosion (AE), the ovaries from dPPCS 1/1 females were small compared to wild-type (wt) ovaries and mutant ovaries did not contain mature eggs ( Fig.  1Aa-b). In wt, the oldest egg chambers found in newly eclosed females are at stage 7, and upon food intake, hormones are produced which trigger the egg chambers to proceed into vitellogenesis, a process whereby the oocyte accumulates nutrients and increases in size [13]. At 72 h AE, 100% (n = 35) of the wt ovaries contained vitellogenic egg chambers, while only 11% of the dPPCS 1/1 ovaries (n = 36) contained vitellogenic egg chambers (Fig. 1Ac-d).
At 120 h AE, 80% of the dPPCS 1/1 ovaries (n = 26) contained vitellogenic egg chambers; however, the two lobes were frequently different in size and displayed features of degenerating egg chambers (Fig. 1Ae).
Between 144-192 h AE, dPPCS 1/1 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 [13]. A Tunnel assay was performed, which revealed that in dPPCS 1/1 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 dPPCS 1/1 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 dPPCS 1/1 background, 11% (n = 166) of the ovariols displayed defects, demonstrating that dPPCS is required for early egg chamber development. Within dPPCS 1/1 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 dPPCS 1/1 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
In addition to impaired fecundity, 80% of the eggs deposited by dPPCS 1/1 females displayed a dumpless phenotype [14] and a wide array of chorion patterning defects (Fig.   1B). Since patterning defects can arise from aberrant actin fiber formation within the nurse cells [15,16], we analyzed actin formation during cytoplasmic dumping. In stage 10 wt egg chambers, an elaborate network of F-actin bundles is assembled inside the nurse cells which is a prelude to cytoplasmic dumping. These bundles anchor the nurse cell nuclei to prevent them from entering the oocyte when the remaining nurse cell material is actively squeezed into the oocyte [17]. Assembly of this F-actin network requires the Quail protein, which colocalizes with the F-actin fibers ( Fig. 2A) [16,18]. In dPPCS 1/1 egg chambers, assembly of the cytoplasmic F-actin fibers was disrupted, and the Quail protein failed to associate with the F-actin bundles and remained diffuse throughout the nurse cell cytoplasm (Fig. 2B). As a result of aberrant Factin assembly, nurse cell nuclei were trapped inside ring canals during dumping (Fig. 2D, Table 1). Interestingly, we also found oocyte nuclei that were encapsulated by bundles of actin (Fig. 2E, Table 1). Furthermore, large actin fibers were assembled at the cortical membrane of the oocyte, and the follicular epithelium of the oocytes was frequently disorganized (Fig. 2H-I). Mutant oocytes also contained large clumps of F-actin ( Fig. 2H-I, Table 1) and we frequently found nurse cell nuclei inside the oocyte compartment (Fig. 2F, J, Table 1).
We stained freshly dissected ovaries with Nile red, which has fluorescent properties in the presence of triacylglycerol and sterol esters [19], to determine if neutral lipid synthesis and transport of these lipids to the oocyte was disrupted during cytoplasmic dumping. In wt, synthesis of these neutral lipids increases in the germ line and somatic cells when egg chambers proceed into late stage oogenesis, and these neutral lipids are transported to the oocyte where they accumulate uniformly near the oocyte membrane (Fig. 3A). In dPPCS 1/1 egg chambers, neutral lipid synthesis was reduced compared to wt, suggesting that the synthesis of neutral lipids is affected in dPPCS mutants (Fig. 3B). Furthermore, accumulation inside the oocyte of these lipids appeared abnormal compared to wt ovaries (compare Fig. 3A and 3B).
Next, we investigated whether a mutation in dPPCS 1/1 affects cell migration events due to defective F-actin organization. During stages 8-10, the border cells, which include the anterior polar cells and part of the main body epithelium, migrate through the nurse cell compartment towards the anterior end of the oocyte [20]. In wt, when the border cells reach the oocyte and the centripetal follicle cells start migrating, Fasciclin III (FasIII) is expressed in the follicle cells of the dorsoanterior corner ( Fig 4A). After centripetal follicle cells finished their migration, FasIII expressing cells form two distinct cell populations at the dorsoanterior surface of the oocyte. Here, formation of the dorsal appendages is initiated (Fig 4B) [14]. In dPPCS 1/1 egg chambers, centripetal migration was finished before the border cells reached the anterior of the oocyte (Fig 4C), indicating that these two cell migration events are not properly synchronized. Together, these data demonstrate that dPPCS is required for F-actin organization and cell migration events during oogenesis.

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 dPPCS 1/1 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) [22]. In dPPCS 1/1 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 dPPCS 1/1 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)P 2 is impaired in
The levels of phospholipids are reduced in dPPCS mutant flies, indicating a general defect in phospholipid biosynthesis [8]. Therefore, it is plausible to assume that phosphatidylinositol (PtdIns) production, the precursor for all phosphoinositides [23], 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 [24].
To investigate whether PtdIns signaling was affected in dPPCS mutant ovaries, we expressed a PLCδ-PH-GFP fusion protein, which is able to bind to PtdIns(4,5)P 2 [25]. We used an Act5C-GAL4 driver to analyze PLCδ-PH-GFP expression and thus PtdIns(4,5)P 2 localization in all cells. During wt cytoplasmic dumping, PtdIns(4,5)P 2 is abundant at the cell membranes of the border cells and the apical membranes of the follicle cells that encapsulate  the oocyte, while low levels of PtdIns(4,5)P 2 can be detected at the nurse cell membranes (Fig. 5A,B,E). In contrast, PtdIns(4,5)P 2 localization at the apical membranes of the follicle cells that encapsulate the oocyte was hardly detectable or absent in dPPCS 1/1 egg chambers (Fig.  5C,D,F,H). Moreover, large patches of follicle cells that encapsulate the oocyte did not accumulate PtdIns(4,5)P 2 at their membranes (Fig. 5C,D,F). Because aberrant apical localization of PtdIns(4,5)P 2 at the follicle cell membranes coincides with impaired oocyte cortex integrity and abnormal F-actin organization (Fig. 5H), this suggests that altered PtdIns(4,5)P 2 signaling could underlie the Factin defects in dPPCS 1/1 egg chambers.
Although the F-actin/PtdIns(4,5)P 2 connection should be investigated in more detail, we propose that F-actin remodeling within the Drosophila ovary likely depends on PtdIns(4,5)P 2 signaling and that this lipid derived signaling route is disrupted in dPPCS 1/1 . Abnormal cytoskeletal organization in dPPCS 1/1 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 dPPCS 1/1 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, dPPCS 1/1 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 dPPCS 1/1 , 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 [11]. 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.

Competing interests
The authors declare that they have no competing interests.    PtdIns(4,5)P 2 localization and expression is affected in dPPCS 1/1 Figure 5 PtdIns(4,5)P 2 localization and expression is affected in dPPCS 1/1 . We overexpressed a PLCδ-PH-GFP fusion protein [25] under the control of a ubiquitously expressed Act5C-GAL4 driver to analyze PtdIns(4,5)P 2 localization and expression in a wt (A,B,E,G) and dPPCS 1