BACKGROUND
embryogenesis in drosophila melanogaster commences with  <dig> nuclear divisions in the absence of cytokinesis, generating a syncytium of ~ <dig>  nuclei located immediately beneath the plasma membrane.
cellularization then ensues, resulting in the synchronous formation of lateral and then basal membranes around each nucleus through rearrangement of the actin cytoskeleton and extensive formation of de novo membrane.
it is estimated that a 25-fold increase in plasma membrane is necessary to complete the process.
the de novo membrane comes from the resorption of microvilli on the outer surface of the blastoderm  <cit>  and the incorporation of er- and/or golgi-derived secretory vesicles  <cit> .
genetic screens have identified numerous proteins that regulate cytoskeletal reorganization during cellularization, several proteins of unknown function including slam  <cit>  that are required at the leading edge of the extending lateral membrane , and a plasma-membrane associated component of the exocytic membrane trafficking machinery, the snare protein syntaxin <dig> .
both syx1a and slam enable the fusion of incoming membrane vesicles into the expanding membranes.
in the absence of slam, membrane vesicles accumulate in the apical cytoplasm.
however, the accumulating vesicles contain rab <dig>  a marker for recycling endosomes, suggesting that the incoming golgi-derived secretory vesicles either first fuse into the apical plasma membrane, and then endocytosis and traffic through the recycling endosomal compartment prior to fusing into the expanding lateral membranes, or traffic directly to the lateral membranes via the rab <dig> endosomal pathway  <cit> .
little is known about the nature of the incoming golgi-derived secretory vesicles or how their apical trafficking is regulated, aside from the findings that lava lamp , a microfilament/microtubule-associated protein that potentially couples the golgi to the microtubule network  <cit> , and the regulated small gtpase rho  <cit>  which is known for its role in cytoskeletal reorganization and membrane vesicle trafficking, are required for the progression of cellularization.
disruption or brefeldin a-mediated inhibiton of exchange factors for the small gtpase arf <dig> also inhibits cellularization  <cit> .
a protein that facilitates golgi-derived vesicle production, trafficking, and fusion into target membranes in yeast and mammalian cells is the signaling enzyme phospholipase d , which hydrolyzes the membrane phospholipid phosphatidylcholine to yield the lipid second messenger phosphatidic acid .
pa is a pleiotropic lipid that functions in membrane vesicle trafficking by promoting membrane budding from the golgi complex  <cit> , by facilitating exocytic trafficking  <cit> , and by promoting vesicle fusion into target membranes  <cit> .
in addition, through regulation of phosphatidylinositol  <dig> -bisphosphate  production, pld/pa regulates actin cytoskeleton reorganization  <cit> .
isolation of plds from yeast and mammals led to the identification of an evolutionarily conserved family of genes  <cit> .
two mammalian pld genes with approximately 50% identity exist, but only a single gene is present in yeast.
mammalian pld activity is regulated by signal transducing pathways in a complex manner involving protein kinase c , rho, and arf, and in turn activates its own effector pathways .
lacking null mutants in animals, current knowledge concerning cellular roles for pld in higher eukaryotes had largely come from overexpression or pharmacological inhibitor experiments in tissue culture systems.
we have recently described the first such genetic model, using drosophila that lack a functional pld gene, and a role for pld in phototransduction, a process that involves both signaling and vesicle trafficking  <cit> .
membrane trafficking as a means to expand or build specialized membranes is a common event in all animals, including during cytokinesis and neurite extension.
we describe here a role for pld in the cellularization pathway that generates and delivers golgi-derived membrane vesicles to the embryonic cortex.
RESULTS
a single phospholipase d  exists in drosophila
our initial report on the existence of a pld gene family  <cit>  described a drosophila sequence-tagged site that encoded a protein fragment with similarity to human pld <dig>  a combination of genomic pcr, conventional screening of ovary cdna libraries, and race-pcr was used to obtain drosophila pld cdnas containing a complete open reading frame .
no other pld-like sequences are present in the bdgp databases, and southern blot analysis using pld as a probe under reduced stringency conditions did not identify additional hybridizing bands , suggesting that there is only one pld gene in drosophila.
the larva and pupa, adult head, and ovary pld cdna clones present in the bdgp database all code for the same amino acid sequence but vary in their 5'utrs .
5'utr usage did not correlate with specific developmental stages or tissue sources.
pld encodes a  <dig> amino acid protein that exhibits ~35% sequence similarity to human pld <dig> , and slightly less to human pld <dig>  the protein contains the conserved regions characteristic of other members of the eukaryotic pld family: a phox homology  domain followed by a pleckstrin homology  domain, conserved catalytic domains , a loop sequence, a pip2-interacting motif, and a conserved carboxy terminus .
the pld amino-terminus is noteworthy for a non-conserved "acidic patch" , whereas the px domain, which in the mammalian isoforms regulates subcellular localization  <cit> , contains an unusual "basic patch" .
px domain functioning in pld may be regulated uniquely by interactions between the acidic and basic patches.
although the central loop region in pld is in the same position as the loop found in mammalian pld <dig>  it lacks similarity to it, suggesting that this region may serve as a hinge region between the two catalytic half-domains, as has been shown for the mammalian pld enzymes  <cit> , rather than to convey structural or functional information.
taken together, the presence of the characteristic pld domains suggested that pld should be a bona fide pld homologue.
pld is activated by components of signaling pathways that are important in cellularization
although yeast pld is constitutively active , mammalian pld is regulated by arf and rho gtpases.
accordingly, the report that pld activity in drosophila could be stimulated by gtpγs  <cit>  suggested that drosophila pld might be regulated similarly.
to investigate this, pld, and a putatively inactive allele of pld containing a point mutation  in the second hkd domain that renders all other known pld proteins inactive  <cit> , were expressed in mammalian cells.
mammalian pld <dig> exhibits little activity in the absence of cellular stimulation  <cit> .
pma, a protein kinase c agonist that activates pld <dig> through both direct and indirect pathways, potently increases pld activity.
in contrast, only a small increase in activity, reflecting activation of endogenous pld, is observed when inactive pld <dig>  is overexpressed and assayed  <cit> .
similarly, drosophila pld activity, but not pld-h1095n activity, significantly increased following pma stimulation .
to determine whether arf and rho stimulate drosophila pld, we used a cell-based system in which increased levels of the gtpases exaggerate mammalian pld responses to g-protein receptor-coupled signaling.
hek <dig> cells expressing the m <dig> muscarinic acetylcholine receptor  <cit>  were co-transfected with pcgn:pld and mammalian arf <dig>  arf <dig>  or rhoa expression plasmids .
a small amount of activity was observed in cells overexpressing the inactive pld-h1095n protein, which derived from the endogenous mammalian plds present in the hek <dig> cells.
similar levels of activity are observed after transfection of inactive human pld <dig>  or empty vectors .
wild-type drosophila pld was activated by receptor signaling through carbachol stimulation and this was enhanced by overexpression of arf <dig>  as has been observed for mammalian pld <dig> .
in contrast, hpld <dig> activity was enhanced more readily by overexpression of rhoa.
taken together, these findings demonstrate that pld is a bona fide pld and can be regulated by arf gtpases, which mediate vesicle trafficking and actin cytoskeleton reorganization in many settings.
pld is maternally and zygotically expressed and peaks prior to cellularization
many of the proteins required for cellularization are synthesized from maternal transcripts, although a few are transcribed zygotically and peak just prior to or at the onset of cellularization  <cit> .
to determine the temporal sequence of pld mrna expression in early embryogenesis, we used quantitative rt-pcr .
pld is expressed maternally, since it was observed in the non-fertilized  0– <dig> hr after egg deposition  collection.
it is also expressed zygotically, based on the dramatic increase observed when zygotic transcription initiates, i.e.
between the fertilized 0– <dig> hr and the  <dig> hr aed collection.
pld mrna levels plummeted between  <dig> and  <dig> hours aed and increased again modestly around  <dig> hours aed.
thus, the temporal pattern of pld expression is consistent with that of genes that have roles in cellularization.
pld localizes to small cytoplasmic vesicles during cellularization
in some mammalian cell types, pld <dig> functions at sites of exocytosis on the plasma membrane to promote the fusion of secretory granules  <cit> .
in others, though, pld <dig> localizes to the golgi and/or peri-nuclear vesicles and facilitates budding from the trans-golgi to generate golgi-derived secretory vesicles , inter-endosomal or er-to-golgi trafficking  <cit> , or trafficking of secretory vesicles to the plasma membrane  <cit> .
determining where pld localizes would therefore suggest potential roles for its function during cellularization.
in cellularizing embryos, pld localized to the cytoplasm and/or to cytosolic vesicles of uniform small size evenly distributed between apical and basal regions of the blastoderm .
pld was not observed to localize specifically to the plasma membrane, membrane furrows, or extending lateral membranes where membrane vesicle fusion takes place.
detection of pld overexpressed by the ngt <dig> promoter  confirmed that the antisera recognizes pld protein.
when overexpressed, pld exhibits a preferential localization for the apical region of the cell; the reason for this is not known but is suggestive with respect to the overexpression phenotype discussed below.
pld also did not co-localize with rab <dig> as a marker for recycling endosomes  or the 120-kd golgi integral membrane protein, p <dig> .
during early cellularization, pld partially co-localized with actin at the sites where cytoskeletal reorganization and membrane formation are taking place .
this overlap ceased by the end of cellularization, as indicated by the clearing of pld within the established actin tracks .
taken together, this pattern of localization would be consistent with a role for pld in translocation of fusion-competent golgi-derived vesicles to the embryonic cortex or regulation of the actin cytoskeleton, although roles in budding or fusion are not ruled out.
to compare the localization of drosophila pld to its mammalian homologs, drosophila pld was expressed in  cos <dig> cells.
pld localized to cytoplasmic vesicles of varying size and to the plasma membrane .
the vesicular staining did not co-localize with the er-marker calreticulin or the recycling endosome proteins rab <dig> and the transferrin receptor .
the pattern of subcellular localization of dpld was intermediate between that of mammalian pld <dig> and pld2: pld <dig> localized to peri-nuclear vesicles, whereas pld <dig> localized to the plasma membrane.
the unique aspects of the drosophila pld pattern of localization could ensue from the non-conserved regions within its amino-terminus and/or loop region.
pld overexpression during early embryogenesis reduces viability
the effect of increasing pld activity during cellularization was examined using the p{gal4-nos.ngt} transgenic binary expression system  <cit>  to deliver wild-type or catalytically-inactive  pld at the start of embryogenesis.
immunostaining indicated that both isoforms were expressed at elevated levels, particularly in the apical region of the newly forming cells .
pld overexpression did not affect golgi localization or morphology .
embryonic lethality averaging  <dig> %  was observed for all p{uas-pld} transgenic lines driven by the strong gal <dig> driver p{gal4-nos.ngt}40; p{gal4-nos.ngt}a. embryonic lethality was dependent on expression level since no lethality was observed with the weaker p{gal4-nos.ngt} <dig> driver.
in addition, lethality was not observed for the strong gal4-driven overexpression of the catalytically-inactive p{uas-pld-h1095n} transgene, indicating that the phenotype ensues from increased pld activity and thus pa production, rather than from a non-enzymatic consequence of pld overexpression.
genetic ablation of pld reduces viability in early embryogenesis
the insertional, or ends-in, homologous recombination method  <cit>  was used to mutate the pld gene.
for gene targeting , a  <dig> -kb genomic fragment of pld beginning in exon  <dig> at amino acid  <dig> of the orf and terminating  <dig> amino acids before the stop codon in exon  <dig> was engineered to carry a recognition site for the i-scei endonuclease and two stop codons within exon  <dig>  seven independent pld targeting events were recovered after screening  <dig>  flies.
of the  <dig> events, only  <dig> had the expected genomic locus organization  and the presence of the two introduced stop codons.
the remaining  <dig> repaired the inserted dna sufficiently distal to the insertion site that one or both stop codons were converted back to the wild-type sequence.
the recombination event generated a tandem duplication of the pld locus resulting in two truncated  pld genes.
analysis of pld mrna from pldnull mutant flies by rt-pcr indicated that only the first gene  was expressed, and trans-splicing did not occur .
the corresponding truncated pld protein generated would be  <dig> amino acids long and contain only  <dig> hkd domain, and thus would be inactive, since as demonstrated previously  <cit> , two functional hkd domains are necessary for pld activity.
expression of this truncated protein should not result in phenotypic consequences, since, as described above, overexpression of a presumptively inactive pld allele ) did not increase pld activity in tissue culture systems and did not cause embryonic lethality.
pldnull flies could be recovered as homozygotes, but displayed a 56% ± 6% reduction in expected viability in comparison to heterozygous flies as determined by mating heterozygous null flies .
this result indicates that zygotic transcription of pld is important, since the maternal pld mrna was present.
a larger decrease in viability  was observed when homozygous null females, collected from the heterozygous stock, were crossed with heterozygous males , indicating that maternal pld is also important.
it should be noted, however, that the effects on viability progressively decreased after maintaining the line for several generations as a homozygous pldnull stock , suggesting that compensatory mechanisms that offset the loss of pld become activated or selected for.
to determine the embryonic developmental stage at which the pldnull flies were arresting, we monitored the progressive development of living pldnull embryos.
of  <dig> y1w67c <dig> control embryos, almost all progressed normally through cellularization and gastrulation .
of  <dig> pldnull embryos, almost half  exhibited visible morphological defects prior to or during cellularization, and  <dig> displayed abnormal gastrulation .
however, it should be noted that of the  <dig> that exhibited abnormal gastrulation ,  <dig> were still able to progress to the larval stage and hatch.
taken together, these results indicate that pld facilitates the normal progression through early embryogenesis.
pld deficiency results in golgi and/or golgi-derived membrane vesicles increased in size
to examine potential mechanisms responsible for the abnormal progression of pldnull embryos during embryogenesis, we assessed the subcellular localization of lava lamp , a golgi-associated protein essential for cellularization that facilitates interaction between golgi secretory vesicles and the cytoskeleton  <cit> .
in wild-type cellularizing embryos, lva localized to golgi vesicles, particularly in basal regions of the forming cells .
in contrast, pldnull embryos exhibited increased fluorescence intensity  upon staining with anti-lva antibody, and the lva-stained golgi vesicles were increased in size .
this observation of abnormal golgi structure suggests a role for pld in facilitating the golgi fissioning required to generate the vesicles destined to be transported via lva to the embryonic cortex.
pld deficiency results in accumulation of neurotactin-containing vesicles at or near the plasma membrane
the transmembrane protein neurotactin , which is synthesized de novo during cellularization, traffics through the secretory pathway and translocates to the apical plasma membrane via golgi-derived vesicles, following which it undergoes endocytosis and re-fusion into lateral membranes via the recycling endosomal pathway.
vesicles bound for lateral membranes may also travel there via the rab <dig> endosomal pathway without first trafficking to the plasma membrane  <cit> .
in pld-deficient embryos, nrt-containing vesicles were observed to accumulate throughout the apical cytoplasm and in a large subapical/apicolateral compartment, in large part juxtaposed to the plasma membrane .
a similar appearing pattern of mis-localization is observed in shibire mutant embryos  <cit> ; in this setting, the aggregating vesicles co-localize with rab <dig>  suggesting that they are composed of recycling endosomes unable to fuse into lateral membranes.
shibire is a dynamin isoform required for budding from recycling endosomes.
however, the aggregating vesicles in pld-deficient embryos did not co-localize with rab <dig> , nor were the rab11-containing vesicles observed to be altered significantly in localization or morphology .
moreover, the nrt vesicle aggregates did not co-localize with a marker for golgi .
thus, nrt appears to exit the golgi into secretory vesicles and translocate apically, but then is not inserted into the plasma membrane, thereby suggesting that, in addition to its role in golgi vesicle fission, pld mediates vesicle fusion at the plasma membrane during cellularization.
as another possibility, pld may mediate fusion of post-rab <dig> vesicles into the lateral membrane.
cellularization is delayed in pldnull embryos
since the pldnull mutants displayed cellularization defects consisting of altered golgi structure and post-golgi trafficking, we examined whether these defects were detrimental for cellularization.
single embryos from an outcrossed pldnull stock were analyzed using video microscopy starting at pole cell formation through the end of cellularization.
three of the six pldnull embryos exhibited a substantial delay during cellularization , taking an average of 28% longer to complete the process , and a fourth was significantly delayed.
two of the six pldnull embryos were wild-type in their progression through cellularization, suggesting incomplete penetrance of the phenotype.
nonetheless, the delay in cellularization in the majority of pld-deficient embryos indicates that the presence of pld facilitates the progression of cellularization in a timely manner.
the embryonic development of single embryos from the time of pole cell formation to the start of gastrulation was recorded using a stereomicroscope with an attached video recorder.
the videos were analyzed from the start of cellularization  to the beginning of pole cell migration and gastrulation.
pldnull embryos were collected from cages set up with homozygous pldnull males and females from an outcrossed heterozygous pldnull stock.
discussion and
CONCLUSIONS
the role of lipid signaling enzymes in the regulation of membrane trafficking during cellularization is largely unknown.
we report here that phospholipase d plays an important role in drosophila embryonic cellularization, using the experimental approaches of overexpression and gene targeting.
the severity of the gene targeting phenotype is potentially understated since pldnull flies appear to activate compensatory mechanisms: heterozygous null flies, when first crossed, generate few homozygous offspring , but continued breeding of the surviving null flies yields a stock with wild-type levels of viability.
such compensation has been observed for lipid signaling  and in general in other drosophila systems  <cit> ).
supporting the possibility of compensatory mechanisms, preliminary experiments using pld rnai on wild-type embryos, a setting in which compensatory mechanisms would not easily be activated, elicited in a nearly complete  inhibition of cellularization .
although there is only one pld isoform in drosophila, which rules out redundant coverage via a closely-related family member, pa levels could be supported through at least three other mechanisms – increasing activity of dag kinase , decreasing activity of phosphatidic phosphohydrolases , or increasing activity of lysophosphatidic acetyl transferases .
a question for the future will be to determine if any of the multiple members of these large gene families are altered in expression in pld-deficient flies.
only recently has the importance of targeted membrane secretion in cellularization been recognized.
lecuit and wieschaus  showed that membrane growth during cellularization occurs in large part through the regulated mobilization of er- and/or golgi-derived membrane populations that insert into precise locations along the plasma membrane.
we report here that on a gross level, loss of pld impairs progression through cellularization.
presumably underlying this observation, two consequences of pld deficiency on vesicle trafficking were observed: altered morphology of the golgi apparatus, from which secretory vesicles are derived, and diminished insertion of the secretory vesicles into the apical and apicolateral plasma membrane.
pld has been reported to play roles in vesicle trafficking in other organisms.
in yeast, pld is required to form the prospore membrane during meiosis via fusion of secretory pathway-derived vesicles  <cit>  and for the generation of secretory vesicles in mutant strains lacking sec14p  <cit> .
in mammalian cells, pld <dig> facilitates secretory vesicle generation  <cit>  and fusion into the plasma membrane  <cit> , whereas pld <dig> has been proposed to facilitate endocytosis  <cit> .
the results we describe here would be consistent with the roles reported for pld <dig> and for yeast pld, and establish the first animal model that can be used to explore the function of pld in vivo.
we have described one other use of this mode, in the exploration of the role of pld in phototransduction  <cit> .
of note, pld does not exhibit steady-state localization in the golgi.
however, studies on mammalian pld have suggested that both pld <dig> and pld <dig> cycle through a succession of subcellular membrane locations including the plasma membrane, recycling and outward bound endosomes, and the golgi, and that they undertake functional roles at multiple steps during the transit  <cit> .
the steady-state pattern of localization reflects just the slowest step in the cycle, which differs in different cell types or as a consequence of the initiation of signaling events.
our observation that pld exhibits both vesicular and plasma membrane localization in mammalian cells  suggests that it too is likely to function at multiple subcellular sites under appropriate circumstances.
future questions include how pld is activated during cellularization and how alterations in pld activity change the morphological structure of the golgi.
numerous questions remain as to how fusion-competent vesicles are generated from the golgi and undergo translocation to sites of incorporation into the extending membranes.
further exploration of the role of pld in this process should lead to new insights.

 embryogenesis in drosophila melanogaster commences with nuclear divisions in the absence of cytokinesis, generating a syncytium of ~ nuclei located immediately beneath the plasma membrane. cellularization then ensues, resulting in the synchronous formation of lateral and then basal membranes around each nucleus through rearrangement of the actin cytoskeleton and extensive formation of de novo membrane. it is estimated that a 25-fold increase in plasma membrane is necessary to complete the process. the de novo membrane comes from the resorption of microvilli on the outer surface of the blastoderm and the incorporation of er- and/or golgi-derived secretory vesicles. genetic screens have identified numerous proteins that regulate cytoskeletal reorganization during cellularization, several proteins of unknown function including slam that are required at the leading edge of the extending lateral membrane, and a plasma-membrane associated component of the exocytic membrane trafficking machinery, the snare protein syntaxin. both syx1a and slam enable the fusion of incoming membrane vesicles into the expanding membranes. in the absence of slam, membrane vesicles accumulate in the apical cytoplasm. however, the accumulating vesicles contain rab a marker for recycling endosomes, suggesting that the incoming golgi-derived secretory vesicles either first fuse into the apical plasma membrane, and then endocytosis and traffic through the recycling endosomal compartment prior to fusing into the expanding lateral membranes, or traffic directly to the lateral membranes via the rab endosomal pathway. little is known about the nature of the incoming golgi-derived secretory vesicles or how their apical trafficking is regulated, aside from the findings that lava lamp, a microfilament/microtubule-associated protein that potentially couples the golgi to the microtubule network, and the regulated small gtpase rho which is known for its role in cytoskeletal reorganization and membrane vesicle trafficking, are required for the progression of cellularization. disruption or brefeldin a-mediated inhibiton of exchange factors for the small gtpase arf also inhibits cellularization. a protein that facilitates golgi-derived vesicle production, trafficking, and fusion into target membranes in yeast and mammalian cells is the signaling enzyme phospholipase d, which hydrolyzes the membrane phospholipid phosphatidylcholine to yield the lipid second messenger phosphatidic acid. pa is a pleiotropic lipid that functions in membrane vesicle trafficking by promoting membrane budding from the golgi complex, by facilitating exocytic trafficking, and by promoting vesicle fusion into target membranes. in addition, through regulation of phosphatidylinositol -bisphosphate production, pld/pa regulates actin cytoskeleton reorganization. isolation of plds from yeast and mammals led to the identification of an evolutionarily conserved family of genes. two mammalian pld genes with approximately 50% identity exist, but only a single gene is present in yeast. mammalian pld activity is regulated by signal transducing pathways in a complex manner involving protein kinase c, rho, and arf, and in turn activates its own effector pathways. lacking null mutants in animals, current knowledge concerning cellular roles for pld in higher eukaryotes had largely come from overexpression or pharmacological inhibitor experiments in tissue culture systems. we have recently described the first such genetic model, using drosophila that lack a functional pld gene, and a role for pld in phototransduction, a process that involves both signaling and vesicle trafficking. membrane trafficking as a means to expand or build specialized membranes is a common event in all animals, including during cytokinesis and neurite extension. we describe here a role for pld in the cellularization pathway that generates and delivers golgi-derived membrane vesicles to the embryonic cortex.  a single phospholipase d exists in drosophila our initial report on the existence of a pld gene family described a drosophila sequence-tagged site that encoded a protein fragment with similarity to human pld a combination of genomic pcr, conventional screening of ovary cdna libraries, and race-pcr was used to obtain drosophila pld cdnas containing a complete open reading frame. no other pld-like sequences are present in the bdgp databases, and southern blot analysis using pld as a probe under reduced stringency conditions did not identify additional hybridizing bands, suggesting that there is only one pld gene in drosophila. the larva and pupa, adult head, and ovary pld cdna clones present in the bdgp database all code for the same amino acid sequence but vary in their 5'utrs. 5'utr usage did not correlate with specific developmental stages or tissue sources. pld encodes a amino acid protein that exhibits ~35% sequence similarity to human pld, and slightly less to human pld the protein contains the conserved regions characteristic of other members of the eukaryotic pld family: a phox homology domain followed by a pleckstrin homology domain, conserved catalytic domains, a loop sequence, a pip2-interacting motif, and a conserved carboxy terminus. the pld amino-terminus is noteworthy for a non-conserved "acidic patch", whereas the px domain, which in the mammalian isoforms regulates subcellular localization, contains an unusual "basic patch". px domain functioning in pld may be regulated uniquely by interactions between the acidic and basic patches. although the central loop region in pld is in the same position as the loop found in mammalian pld it lacks similarity to it, suggesting that this region may serve as a hinge region between the two catalytic half-domains, as has been shown for the mammalian pld enzymes, rather than to convey structural or functional information. taken together, the presence of the characteristic pld domains suggested that pld should be a bona fide pld homologue. pld is activated by components of signaling pathways that are important in cellularization although yeast pld is constitutively active, mammalian pld is regulated by arf and rho gtpases. accordingly, the report that pld activity in drosophila could be stimulated by gtpγs suggested that drosophila pld might be regulated similarly. to investigate this, pld, and a putatively inactive allele of pld containing a point mutation in the second hkd domain that renders all other known pld proteins inactive, were expressed in mammalian cells. mammalian pld exhibits little activity in the absence of cellular stimulation. pma, a protein kinase c agonist that activates pld through both direct and indirect pathways, potently increases pld activity. in contrast, only a small increase in activity, reflecting activation of endogenous pld, is observed when inactive pld is overexpressed and assayed. similarly, drosophila pld activity, but not pld-h1095n activity, significantly increased following pma stimulation. to determine whether arf and rho stimulate drosophila pld, we used a cell-based system in which increased levels of the gtpases exaggerate mammalian pld responses to g-protein receptor-coupled signaling. hek cells expressing the m muscarinic acetylcholine receptor were co-transfected with pcgn:pld and mammalian arf arf or rhoa expression plasmids. a small amount of activity was observed in cells overexpressing the inactive pld-h1095n protein, which derived from the endogenous mammalian plds present in the hek cells. similar levels of activity are observed after transfection of inactive human pld or empty vectors. wild-type drosophila pld was activated by receptor signaling through carbachol stimulation and this was enhanced by overexpression of arf as has been observed for mammalian pld. in contrast, hpld activity was enhanced more readily by overexpression of rhoa. taken together, these findings demonstrate that pld is a bona fide pld and can be regulated by arf gtpases, which mediate vesicle trafficking and actin cytoskeleton reorganization in many settings. pld is maternally and zygotically expressed and peaks prior to cellularization many of the proteins required for cellularization are synthesized from maternal transcripts, although a few are transcribed zygotically and peak just prior to or at the onset of cellularization. to determine the temporal sequence of pld mrna expression in early embryogenesis, we used quantitative rt-pcr. pld is expressed maternally, since it was observed in the non-fertilized 0– hr after egg deposition collection. it is also expressed zygotically, based on the dramatic increase observed when zygotic transcription initiates, i.e. between the fertilized 0– hr and the hr aed collection. pld mrna levels plummeted between and hours aed and increased again modestly around hours aed. thus, the temporal pattern of pld expression is consistent with that of genes that have roles in cellularization. pld localizes to small cytoplasmic vesicles during cellularization in some mammalian cell types, pld functions at sites of exocytosis on the plasma membrane to promote the fusion of secretory granules. in others, though, pld localizes to the golgi and/or peri-nuclear vesicles and facilitates budding from the trans-golgi to generate golgi-derived secretory vesicles, inter-endosomal or er-to-golgi trafficking, or trafficking of secretory vesicles to the plasma membrane. determining where pld localizes would therefore suggest potential roles for its function during cellularization. in cellularizing embryos, pld localized to the cytoplasm and/or to cytosolic vesicles of uniform small size evenly distributed between apical and basal regions of the blastoderm. pld was not observed to localize specifically to the plasma membrane, membrane furrows, or extending lateral membranes where membrane vesicle fusion takes place. detection of pld overexpressed by the ngt promoter confirmed that the antisera recognizes pld protein. when overexpressed, pld exhibits a preferential localization for the apical region of the cell; the reason for this is not known but is suggestive with respect to the overexpression phenotype discussed below. pld also did not co-localize with rab as a marker for recycling endosomes or the 120-kd golgi integral membrane protein, p. during early cellularization, pld partially co-localized with actin at the sites where cytoskeletal reorganization and membrane formation are taking place. this overlap ceased by the end of cellularization, as indicated by the clearing of pld within the established actin tracks. taken together, this pattern of localization would be consistent with a role for pld in translocation of fusion-competent golgi-derived vesicles to the embryonic cortex or regulation of the actin cytoskeleton, although roles in budding or fusion are not ruled out. to compare the localization of drosophila pld to its mammalian homologs, drosophila pld was expressed in cos cells. pld localized to cytoplasmic vesicles of varying size and to the plasma membrane. the vesicular staining did not co-localize with the er-marker calreticulin or the recycling endosome proteins rab and the transferrin receptor. the pattern of subcellular localization of dpld was intermediate between that of mammalian pld and pld2: pld localized to peri-nuclear vesicles, whereas pld localized to the plasma membrane. the unique aspects of the drosophila pld pattern of localization could ensue from the non-conserved regions within its amino-terminus and/or loop region. pld overexpression during early embryogenesis reduces viability the effect of increasing pld activity during cellularization was examined using the p{gal4-nos.ngt} transgenic binary expression system to deliver wild-type or catalytically-inactive pld at the start of embryogenesis. immunostaining indicated that both isoforms were expressed at elevated levels, particularly in the apical region of the newly forming cells. pld overexpression did not affect golgi localization or morphology. embryonic lethality averaging % was observed for all p{uas-pld} transgenic lines driven by the strong gal driver p{gal4-nos.ngt}40; p{gal4-nos.ngt}a. embryonic lethality was dependent on expression level since no lethality was observed with the weaker p{gal4-nos.ngt} driver. in addition, lethality was not observed for the strong gal4-driven overexpression of the catalytically-inactive p{uas-pld-h1095n} transgene, indicating that the phenotype ensues from increased pld activity and thus pa production, rather than from a non-enzymatic consequence of pld overexpression. genetic ablation of pld reduces viability in early embryogenesis the insertional, or ends-in, homologous recombination method was used to mutate the pld gene. for gene targeting, a -kb genomic fragment of pld beginning in exon at amino acid of the orf and terminating amino acids before the stop codon in exon was engineered to carry a recognition site for the i-scei endonuclease and two stop codons within exon seven independent pld targeting events were recovered after screening flies. of the events, only had the expected genomic locus organization and the presence of the two introduced stop codons. the remaining repaired the inserted dna sufficiently distal to the insertion site that one or both stop codons were converted back to the wild-type sequence. the recombination event generated a tandem duplication of the pld locus resulting in two truncated pld genes. analysis of pld mrna from pldnull mutant flies by rt-pcr indicated that only the first gene was expressed, and trans-splicing did not occur. the corresponding truncated pld protein generated would be amino acids long and contain only hkd domain, and thus would be inactive, since as demonstrated previously, two functional hkd domains are necessary for pld activity. expression of this truncated protein should not result in phenotypic consequences, since, as described above, overexpression of a presumptively inactive pld allele ) did not increase pld activity in tissue culture systems and did not cause embryonic lethality. pldnull flies could be recovered as homozygotes, but displayed a 56% ± 6% reduction in expected viability in comparison to heterozygous flies as determined by mating heterozygous null flies. this result indicates that zygotic transcription of pld is important, since the maternal pld mrna was present. a larger decrease in viability was observed when homozygous null females, collected from the heterozygous stock, were crossed with heterozygous males, indicating that maternal pld is also important. it should be noted, however, that the effects on viability progressively decreased after maintaining the line for several generations as a homozygous pldnull stock, suggesting that compensatory mechanisms that offset the loss of pld become activated or selected for. to determine the embryonic developmental stage at which the pldnull flies were arresting, we monitored the progressive development of living pldnull embryos. of y1w67c control embryos, almost all progressed normally through cellularization and gastrulation. of pldnull embryos, almost half exhibited visible morphological defects prior to or during cellularization, and displayed abnormal gastrulation. however, it should be noted that of the that exhibited abnormal gastrulation, were still able to progress to the larval stage and hatch. taken together, these results indicate that pld facilitates the normal progression through early embryogenesis. pld deficiency results in golgi and/or golgi-derived membrane vesicles increased in size to examine potential mechanisms responsible for the abnormal progression of pldnull embryos during embryogenesis, we assessed the subcellular localization of lava lamp, a golgi-associated protein essential for cellularization that facilitates interaction between golgi secretory vesicles and the cytoskeleton. in wild-type cellularizing embryos, lva localized to golgi vesicles, particularly in basal regions of the forming cells. in contrast, pldnull embryos exhibited increased fluorescence intensity upon staining with anti-lva antibody, and the lva-stained golgi vesicles were increased in size. this observation of abnormal golgi structure suggests a role for pld in facilitating the golgi fissioning required to generate the vesicles destined to be transported via lva to the embryonic cortex. pld deficiency results in accumulation of neurotactin-containing vesicles at or near the plasma membrane the transmembrane protein neurotactin, which is synthesized de novo during cellularization, traffics through the secretory pathway and translocates to the apical plasma membrane via golgi-derived vesicles, following which it undergoes endocytosis and re-fusion into lateral membranes via the recycling endosomal pathway. vesicles bound for lateral membranes may also travel there via the rab endosomal pathway without first trafficking to the plasma membrane. in pld-deficient embryos, nrt-containing vesicles were observed to accumulate throughout the apical cytoplasm and in a large subapical/apicolateral compartment, in large part juxtaposed to the plasma membrane. a similar appearing pattern of mis-localization is observed in shibire mutant embryos; in this setting, the aggregating vesicles co-localize with rab suggesting that they are composed of recycling endosomes unable to fuse into lateral membranes. shibire is a dynamin isoform required for budding from recycling endosomes. however, the aggregating vesicles in pld-deficient embryos did not co-localize with rab, nor were the rab11-containing vesicles observed to be altered significantly in localization or morphology. moreover, the nrt vesicle aggregates did not co-localize with a marker for golgi. thus, nrt appears to exit the golgi into secretory vesicles and translocate apically, but then is not inserted into the plasma membrane, thereby suggesting that, in addition to its role in golgi vesicle fission, pld mediates vesicle fusion at the plasma membrane during cellularization. as another possibility, pld may mediate fusion of post-rab vesicles into the lateral membrane. cellularization is delayed in pldnull embryos since the pldnull mutants displayed cellularization defects consisting of altered golgi structure and post-golgi trafficking, we examined whether these defects were detrimental for cellularization. single embryos from an outcrossed pldnull stock were analyzed using video microscopy starting at pole cell formation through the end of cellularization. three of the six pldnull embryos exhibited a substantial delay during cellularization, taking an average of 28% longer to complete the process, and a fourth was significantly delayed. two of the six pldnull embryos were wild-type in their progression through cellularization, suggesting incomplete penetrance of the phenotype. nonetheless, the delay in cellularization in the majority of pld-deficient embryos indicates that the presence of pld facilitates the progression of cellularization in a timely manner. the embryonic development of single embryos from the time of pole cell formation to the start of gastrulation was recorded using a stereomicroscope with an attached video recorder. the videos were analyzed from the start of cellularization to the beginning of pole cell migration and gastrulation. pldnull embryos were collected from cages set up with homozygous pldnull males and females from an outcrossed heterozygous pldnull stock. discussion and  the role of lipid signaling enzymes in the regulation of membrane trafficking during cellularization is largely unknown. we report here that phospholipase d plays an important role in drosophila embryonic cellularization, using the experimental approaches of overexpression and gene targeting. the severity of the gene targeting phenotype is potentially understated since pldnull flies appear to activate compensatory mechanisms: heterozygous null flies, when first crossed, generate few homozygous offspring, but continued breeding of the surviving null flies yields a stock with wild-type levels of viability. such compensation has been observed for lipid signaling and in general in other drosophila systems ). supporting the possibility of compensatory mechanisms, preliminary experiments using pld rnai on wild-type embryos, a setting in which compensatory mechanisms would not easily be activated, elicited in a nearly complete inhibition of cellularization. although there is only one pld isoform in drosophila, which rules out redundant coverage via a closely-related family member, pa levels could be supported through at least three other mechanisms – increasing activity of dag kinase, decreasing activity of phosphatidic phosphohydrolases, or increasing activity of lysophosphatidic acetyl transferases. a question for the future will be to determine if any of the multiple members of these large gene families are altered in expression in pld-deficient flies. only recently has the importance of targeted membrane secretion in cellularization been recognized. lecuit and wieschaus showed that membrane growth during cellularization occurs in large part through the regulated mobilization of er- and/or golgi-derived membrane populations that insert into precise locations along the plasma membrane. we report here that on a gross level, loss of pld impairs progression through cellularization. presumably underlying this observation, two consequences of pld deficiency on vesicle trafficking were observed: altered morphology of the golgi apparatus, from which secretory vesicles are derived, and diminished insertion of the secretory vesicles into the apical and apicolateral plasma membrane. pld has been reported to play roles in vesicle trafficking in other organisms. in yeast, pld is required to form the prospore membrane during meiosis via fusion of secretory pathway-derived vesicles and for the generation of secretory vesicles in mutant strains lacking sec14p. in mammalian cells, pld facilitates secretory vesicle generation and fusion into the plasma membrane, whereas pld has been proposed to facilitate endocytosis. the results we describe here would be consistent with the roles reported for pld and for yeast pld, and establish the first animal model that can be used to explore the function of pld in vivo. we have described one other use of this mode, in the exploration of the role of pld in phototransduction. of note, pld does not exhibit steady-state localization in the golgi. however, studies on mammalian pld have suggested that both pld and pld cycle through a succession of subcellular membrane locations including the plasma membrane, recycling and outward bound endosomes, and the golgi, and that they undertake functional roles at multiple steps during the transit. the steady-state pattern of localization reflects just the slowest step in the cycle, which differs in different cell types or as a consequence of the initiation of signaling events. our observation that pld exhibits both vesicular and plasma membrane localization in mammalian cells suggests that it too is likely to function at multiple subcellular sites under appropriate circumstances. future questions include how pld is activated during cellularization and how alterations in pld activity change the morphological structure of the golgi. numerous questions remain as to how fusion-competent vesicles are generated from the golgi and undergo translocation to sites of incorporation into the extending membranes. further exploration of the role of pld in this process should lead to new insights. 
our results suggest that pld functions to promote trafficking of golgi-derived fusion-competent vesicles during cellularization. we describe here involvement of the signaling enzyme phospholipase d in regulation of this developmental step.
 cellularization of the drosophila embryo is an unusually synchronous form of cytokinesis in which polarized membrane extension proceeds in part through incorporation of new membrane via fusion of apically-translocated golgi-derived vesicles.  we describe here involvement of the signaling enzyme phospholipase d in regulation of this developmental step. functional analysis using gene targeting revealed that cellularization is hindered by the loss of pld, resulting frequently in early embryonic developmental arrest. mechanistically, chronic pld deficiency causes abnormal golgi structure and secretory vesicle trafficking.  our results suggest that pld functions to promote trafficking of golgi-derived fusion-competent vesicles during cellularization. 