BACKGROUND
recently characterised manf  and cdnf  form an independent family of neurotrophic factors  <cit> .
manf was identified from a conditioned media of cultured mesencephalic astrocytes in a search for secreted factors supporting dopamine  neurons  <cit> .
specific loss of da neurons is the characteristic feature of parkinson's disease .
therefore factors that maintain and support da neurons are attractive candidates for the treatment of pd.
manf was shown to support the survival of cultured primary da neurons but to have no effect on cultured gabaergic or serotonergic neurons  <cit> .
subsequently, mammalian manf and its paralog cdnf were shown to prevent the loss of da neurons in mouse 6-ohda pd model  <cit> .
contrary to other vertebrate neurotrophic factors the manf/cdnf family is evolutionarily well conserved among multicellular organisms including the fruit fly, drosophila melanogaster  <cit> .
importantly, the protective role of manf for da neurons is also conserved  <cit> .
apparently both mammals and invertebrates share the same signalling partners as the lack of drosophila manf can be substituted by human manf  <cit> .
however, the interaction partners or how these proteins act at the molecular level are still elusive.
it is important to understand the mechanisms of how these manf/cdnf family proteins work at molecular level before the potential therapeutic applications.
recent studies have shown the protective role of mammalian manf  to be more general than restricted to the nervous system.
manf is upregulated by upr in several mammalian cell lines  <cit>  and by ischemia induced upr in the heart and brain  <cit> .
er is the central regulator of protein folding and quality control and it has to adapt its capacity to the specific need of a particular cell type.
conditions challenging the function of the er, like an increase of newly synthesized unfolded proteins in its lumen, lead to upr  <cit> .
in eukaryotes, the three canonical branches of upr are mediated by er membrane-associated sensor proteins.
in stress-free, functional er the intralumenal domains of these sensor proteins are bound to a chaperone bip/grp <dig>  and maintained inactive  <cit> .
the upr intersect with a variety of inflammatory and stress signalling pathways and networks activated by oxidative stress, all of which can influence cell metabolism.
er stress and upr have also been implicated in the pathogenesis of several neurodegenerative diseases because of their characteristic accumulation of specific misfolded proteins  <cit> .
data from pd patients reveal that in da neurons of substantia nigra the upr is activated  <cit> .
recently, drosophila has been used progressively to model human neurodegenerative diseases and upr  <cit> .
we have previously generated and characterized drosophila manf mutants.
the zygotic null mutants  survive up to 2nd instar larvae due to the high maternal contribution.
mutants lacking both maternal and zygotic manf  are late embryonic lethal and never hatch  <cit> .
the embryonic lack of maternal manf gene products and the lethality is rescued by paternal manf gene expression.
both manfmzΔ <dig> and manfΔ <dig> mutants share nervous system defects, particularly dopamine neurites are altered and degenerate.
ectopic overexpression of manf reveals no evident abnormalities in the embryonic or larval nervous system development or in the adult flies .
here we compare the mrna expression profiles of manfmzΔ <dig> mutant embryos, manfΔ <dig> mutant larvae, paternally rescued maternal mutant embryos manfmΔ96/+, and manf ubiquitously overexpressing larvae to the wild type animals of exactly the same stages.
RESULTS
the most prevalent changes in gene expression occur in manf mutants that lack the maternal contribution of manf
for microarray gene expression analysis we used two developmental stages in combination with three separate genotypes.
the age of embryos and larvae were selected according to the lifespan of the manf mutants.
manfmzΔ <dig> mutants fail in tracheal air filling and never hatch.
mutant manfmzΔ <dig> embryos were picked during the late stage of  <dig>  just before hatching when the trachea of wild type embryos fill with air.
mutant manfΔ <dig> larvae with maternal contribution survive to approximately  <dig> hours ael and were collected as first instars 29- <dig> hours ael when maternal loads of manf gene products have decreased.
three biologically independent sets of samples were used for microarray analysis.
the expression profiles of all transgenic and mutant animals were compared to the wild type of the corresponding developmental stage.
the numerical overview of statistically significant differences  showed the most prominent changes in gene expression of manfmzΔ <dig> mutants .
the smallest changes took place in the case of paternal rescue  .
among the differentially regulated genes, approximately half were up- or downregulated in different manf conditions.
altogether we validated  <dig> genes of the microarray results.
genes were selected by several criteria such as differential expression or similar regulation in both mutants or otherwise high representation in the whole dataset.
as a result,  <dig> % of validated genes in different genetic conditions were independently confirmed by qpcr .
the number of differentially regulated probes was compared.
in the agilent drosophila microarray design  there was unequal number of probes targeting the particular gene, ranging from one probe to several per gene.
only statistically significant results  are presented.
results of qpcr were made comparable to microarray fold change  by calculating the log <dig> from the relative fold changes.
t-test means calculated p-value associated with student's t-test.
only statistically significant  results are presented.
results of qpcr results were made comparable to microarray fold change  by calculating the log <dig> of the relative fold changes.
t-test means calculated p-value associated with student's t-test.
membrane transporters and metabolic genes are downregulated in manfmzΔ <dig> mutants
development of maternal and zygotic mutant manfmzΔ <dig> proceeds until stage  <dig> with no differences to wild type embryos, but  <dig> hours ael the cuticle and the nervous system defects become evident  <cit> .
in comparison to wild type embryos of the same age, in manfmzΔ <dig> mutants  <dig> genes were found to be downregulated.
these genes were grouped into  <dig> functional clusters , among which the most significantly enriched clusters were related to membrane transporters  and transmembrane proteins  .
there were several enriched clusters referring to different metabolic processes such as amine, amino acid and carboxylic acid catabolic processes , dna metabolic processes , and genes related to pyrimidine metabolism .
clusters are represented starting from the highest enrichment score in diminishing order.
only the highest clusters are shown, for the full list of david cluster analysis of downregulated genes in manfmzΔ <dig> mutants, see additional file  <dig>  in the case of missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above.
among the downregulated genes in manfmzΔ <dig> mutants, the gene ontology  term of mitochondria-related transcripts was highly enriched .
mitochondria are the respiratory machinery of the cell responsible for oxidation processes and participate in maintaining cellular homeostasis.
the lack of manf causes downregulation of several components in all mitochondrial compartments: the lumen as well as the inner and outer membranes.
stress and defence response related genes are induced in manfmzΔ <dig> mutants
in manfmzΔ <dig> mutant embryos,  <dig> genes were upregulated in comparison to wild type embryos of the same stage .
the most prominent group of significantly enriched go terms was immune and defense response , followed by the groups related to proteolysis, hydrolases and peptidases  .
the upregulated gene set was also enriched in genes related to actin cytoskeleton organization and actin filament-based process .
moreover, genes involved in cell death  were prominently enriched as well.
clusters are represented starting from the highest enrichment score in diminishing order.
only the highest clusters are shown, for the full list of david cluster analysis of upregulated genes in manfmzΔ <dig> mutants, see additional file  <dig>  in the case on missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above.
enzymes for dopamine synthesis are upregulated despite of low dopamine levels
extremely low dopamine levels are detected in manfmzΔ <dig> embryos  <cit> .
nonetheless, we observed significant upregulation of transcripts of the dopamine producing enzymes tyrosine hydroxylase  and dopa decarboxylase  .
punch; encoding a gtp cyclohydrolase, an enzyme for tetrahydrobiopterin  synthesis was also upregulated in manfmzΔ <dig> embryos .
there could be several explanations for these alterations.
tyrosine, the essential amino acid for dopamine synthesis, is transported into the cell from hemolymph.
in manf mutants several amino acid membrane transporters were downregulated.
the lack of substrate, tyrosine, together with low amounts of the end product, dopamine, could lead to the upregulation of the genes coding for the enzymes in dopamine synthesis pathway and their cofactors.
genes involved in nucleic acid metabolism and protein folding are downregulated in larval manfΔ <dig> mutants
larval manfΔ <dig> mutants with maternally contributed manf gene products never reach 3rd instar stage and rarely survive up to  <dig> hours ael at 25°c.
initially, manfΔ <dig> mutant larvae hatch and feed normally.
whereas wild type larvae grow rapidly, the mutant larvae remain smaller and start to wander away from food, become sluggish and stop moving, still responding to touch and usually die during the first larval molt  <cit> .
because of the temporal differences in survival between individual manfΔ <dig> mutant larvae from 1st to 2nd instar, for microarray analysis we collected larvae 29- <dig> hours ael.
when comparing the expression profile of larval manfΔ <dig> mutants to the wild type larvae, almost half the number of genes  was significantly downregulated as compared to the rate in manfmzΔ <dig> embryos resulting in  <dig> functional clusters .
the most enriched go terms fell into clusters related to intracellular organelle lumen and nucleic acid metabolic processes .
the cellular activities such as dna replication, rna processing and splicing were enriched among downregulated genes.
the 5th highly enriched cluster consisted of go terms such as er related genes , proline and arginine metabolism , and oxioreductases .
mitotic cell cycle, chromosomal segregation, and mitotic spindle organization were also clustered as significantly enriched.
these changes could be linked to upr, as one of the outcomes of upr is general and unspecific downregulation of novel protein synthesis, at the same time activating the protein synthesis for chaperones and genes enhancing the protein folding to release the unfolded protein load in er.
clusters are represented starting from the highest enrichment score in diminishing order.
only the highest clusters are shown, for the full list of david cluster analysis of downregulated genes in manfΔ <dig> mutants, see additional file  <dig>  in the case on missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above.
sugar metabolism, hydrolases, and er related oxidation reduction genes are induced in manfΔ <dig> larvae
in manfΔ <dig> larval mutants,  <dig> genes showed upregulation in comparison to the wild type larvae.
the most enriched functional clusters included go terms like sugar metabolism and glucosidases, glycosyl hydrolases , and hydrolases and carboxylesterases , followed by cluster of monooxygenases, cytochrome p <dig>  iron, vesicular fraction, oxidation reduction and endoplasmic reticulum  .
chitin and polysaccharide metabolism was also among the highly enriched go terms .
the 5th ranked cluster of go terms was immune and defence response , which was the most highly enriched cluster in manfmzΔ <dig> mutant embryos.
genes related to rna metabolism, atp binding, and dna replication are downregulated in both manf mutants
next, we looked for functional terms among the  <dig> commonly downregulated genes in both manf mutants .
there was 30% of overlap in gene sets between the manfΔ <dig> and manfmzΔ <dig> mutants.
among the downregulated overlapping genes, the enrichment of go terms fell into rna metabolism and ribosome biogenesis .
around 10% of all known atp binding genes were downregulated  together with  <dig> genes of the purine and pyrimidine metabolism.
additionally, the genes coding sugar transporters and the genes involved in transmembrane transport  highly represented among downregulated genes in manfmzΔ <dig> mutant embryos, were repressed in zygotic mutant manfΔ <dig> as well.
go terms related to dna replication  were also enriched among the commonly downregulated genes in both manf mutants.
altogether, the manfmzΔ <dig> mutant embryos lacking both maternal and zygotic manf showed twice more drastic decline from wild type transcriptome than manfΔ <dig> larval mutants, whose maternal transcripts gradually diminish.
beside behavioural and growth phenotype, manfΔ <dig> larvae show degeneration of dopaminergic neurites in ventral nerve cord  <cit> .
we found three genes downregulated in both mutants that are involved in neurite development: abelson tyrosine kinase , guanine nucleotide exchange factor gef64c  and the transcription factor longitudinals lacking .
a third of all upregulated genes  were induced in both mutants .
immune and defense response was the most enriched functional cluster  along with the group consisting of monooxygenases, oxidoreductases, vesicular fraction, endoplasmic reticulum, cytochrome p <dig> and lipid metabolic process .
controversially, disabled , was upregulated among the  <dig> genes involved in neuronal development e.g.
transcription factor krüppel , negative regulator of growth shrub , insulin receptor , and drosophila extracellular-signal-regulated kinase  rolled .
genes related to upr were upregulated in manf mutants
previous in vitro studies using tunicamycin, the inhibitor of glycosylation, to induce er stress in mammalian cell lines have shown in upr the upregulation of manf  <cit> .
in rat neonatal cardiomyocytes in response to upr manf is secreted to promote cellular survival  <cit> .
er stress and one of the consequences, upr, has been mainly studied in yeast and mammalian cells.
in drosophila, there are several recent studies where upr has been addressed  <cit> .
manf has been shown to be upregulated after feeding tunicamycin to adult fruit flies indicating the involvement of manf in chemically induced upr in drosophila  <cit> .
to find out the intracellular localisation of manf in drosophila, we used larval 2nd instar garland cells.
garland cells are nephrocytes with high rate of endocytosis and express several neuronal and exocytosis markers e.g.
prospero , snare binding protein ras opposite  facilitating neurotransmitter secretion, and syntaxin 1a   <cit> .
these cells have the most abundant expression of manf starting from embryogenesis  <cit> .
in the garland cells, manf was localised around the nucleus, partially overlapping with er-targeted marker .
next, we tested the hypothesis that the metabolic changes in manf mutant could be the result of severe er stress caused by altered expression of er related genes.
drosophila genes homologous to several er stress pathway have been identified.
out of  <dig> genes involved in er and protein processing in the kegg database,  <dig> have one or more homologues in fruit flies .
of these upr related drosophila genes, 30% showed altered gene expression in our microarray experiment.
altogether,  <dig> genes involved in er and protein processing show statistically significant expression changes.
the gene cg <dig> is an annotated gene with unknown function in drosophila.
its human homologue nucleotide exchange factor sil <dig>  is a bip binding protein.
in humans, several mutations in sil <dig> gene disrupting the protein cause the marinesco-sjögren syndrome , an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy  <cit> .
we validated cg <dig> by qpcr as downregulated by manf overexpression and upregulated when manf is abolished in drosophila embryos and larvae.
it has been shown by immunoprecipitation studies that mammalian manf binds to bip  <cit> .
thus it is possible that manf and cg <dig> compete in binding to bip together with unfolded proteins.
as the ectopic overexpression of manf has no effect on fruit fly viability or nervous system development , the diminished transcript level for cg <dig> is not comparable to the total lack of this gene product in the mss patients.
according to our qpcr validated microarray results several other genes implicated in upr were downregulated in larvae overexpressing manf, such as pancreatic eif-2α kinase , heat shock protein  <dig> , ubiquilin , and septin interacting protein  <dig> .
in embryonic manfmzΔ <dig> mutants all above mentioned genes were significantly upregulated as well as considerable number of other er chaperone genes .
furthermore, when evaluating the ultrastructural changes in manfmzΔ <dig> mutants, we noticed that the er was swollen and dilated in epidermal cells, indicating severe disturbances of er structure .
in manfmzΔ <dig> mutant embryos the extent of phosphorylated eukaryotic initiation factor eif2α was more than two fold upregulated when compared to the wild type  indicating the presence of upr in these manf mutants.
the phosphorylation of eif2α by perk is a hallmark for upr, resulting in reversible blockage of translation and downregulation of the protein load to the er  <cit> .
in drosophila there are two kinases, perk and gcn <dig>  shown to be able to phosphorylate eif2α  <cit> .
the expression of gcn <dig> is high only during early stages of embryogenesis  <cit> .
thus perk is a potential candidate kinase behind eif2α phosphorylation at the end of embryogenesis.
interestingly, our microarray data showed that in manfmzΔ <dig> mutants the transcription of perk was  <cit>  upregulated and the genes involved in different metabolic processes such as amino acid, dna and pyrimidine metabolism were downregulated indicating overall inhibition of translation.
so it is probable that the upr perk pathway is activated in manfmzΔ <dig> mutants.
the second upr sensor, ire <dig>  activates two separate downstream branches.
one of the branches leads to the activation of jun kinase and death pathway  <cit> .
according to the microarray results, drosophila jun kinase kinase hemipterous showed significant upregulation in both manf mutants.
significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression.
gene name stands for the particular homologue gene name in drosophila.
notice that the same kegg identifier can lead to several different genes.
p.res = paternal rescue, mut = mutant, o.ex.
= overexpression.
conclusively, the absolute lack of manf results in severe er stress and upregulation of many genes involved in upr finally leading to the cell death.
when maternal manf stores are gradually decreased in manfΔ <dig> larvae, there are only few genes upregulated that are related to er: cg <dig>  ubiquitin protein ligase lin <dig>  heat shock protein cognate  <dig> and ubiquitin-conjugating enzyme cg <dig>  as our data come mostly from gene expression analyses, further biochemical experiments are needed to identify the exact role of manf in upr.
lack of manf results in downregulation of several genes in exocytosis pathway
ultrastructural study of manfmzΔ <dig> mutants revealed overload of vesicles next to the apical part of epidermal cells and reduced microvillae thought to enhance the capacity of the secretion of these cells .
this result, together with the severe defects observed in the cuticle secretion and organisation , suggested a possible involvement of the genes of the exocytosis pathway.
indeed, the expression of several genes related to exocytosis and snare transport were altered in different manf conditions .
in manf mutants, several genes implied in exocytosis and vesicle transport from golgi complex to the plasma membrane were downregulated , whereas the er residing syntaxins - stx <dig> and stx <dig> - were upregulated.
this supports an inhibition of secretion from golgi complex to the plasma membrane as seen in manfmzΔ <dig> mutant epidermal cells in vesicle accumulation close to the apical area .
significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression.
gene name stands for the particular homologue gene name in drosophila.
few related drosophila genes associated with neurotransmission missing from kegg pathway were added.
the according coloured scheme for exocytosis and snare of kegg pathway is presented in additional file  <dig>  p.res = paternal rescue, mut = mutant, o.ex.
= overexpression.
expression of genes involved in cuticle development were altered in manfmzΔ <dig> mutants
we have previously shown that manfmzΔ <dig> embryos have disorganized cuticle  <cit> .
at the end of embryogenesis from stage  <dig> onward, the cuticle components are secreted by epithelial cells and stored in regular layers, and subsequently the cuticular proteins are crosslinked by dopamine-derived quinones  <cit> .
among the downregulated genes in manfmzΔ <dig> embryos, there were  <dig> genes coding the structural components of the insect cuticle.
at the same time, several other genes responsible for cuticle development were upregulated, such as the genes encoding enzymes involved in chitin synthesis, krotzkopf verkehrt   <cit> , knickkopf   <cit> , and syx1a .
additionally, several genes involved in epithelial development and morphogenesis were upregulated and significantly enriched among the go terms  .
we used transmission electron microscopy  analysis in manfmzΔ <dig> mutants at the embryonic stage  <dig> to investigate the epithelial cells responsible for cuticle secretion.
indeed, these cells showed morphologically abnormal er and accumulation of vesicles in the apical part .
it is possible that the enhanced endocytosis and disturbed exocytosis, together with misbalance in cuticular components, lead to disorganised and disrupted cuticle in manfmzΔ <dig> mutant embryos.
in larval manfΔ <dig> mutant with gradually fading maternal contribution, the cuticle showed no disruption and the chitin layers were deposited and organised normally .
instead there were problems in shedding the old cuticle and often the 1st instar cuticle remained attached .
this implies that the maternal loading of manf gene products in larval manfΔ <dig> mutants was sufficient to overcome defects in early cuticle development, secretion and layering, but insufficient to complete the first molt.
large vesicles filled with electron dense debris are accumulated in manfmzΔ <dig> mutant
to investigate the routes of membrane trafficking we evaluated genes involved in endocytosis.
of all drosophila homologues known to be involved in endocytosis, 47% showed significant expression changes in our microarray experiment .
genes coding for components of multivesicular body formation were especially altered.
several transmembrane receptors of growth factors were downregulated in manf mutants and upregulated when manf was overexpressed.
pdgf- and vegf-receptor related pvr was upregulated in larvae in both lack and overexpression of manf.
cbl, an e <dig> ubiquitin ligase and negative regulator of tyrosine kinase receptor signalling, was downregulated in mutant larvae and upregulated under manf overexpression conditions.
two different members of endosomal recycling pathway, par family members and rab-protein  <dig>  were upregulated in mutants.
par transcripts were upregulated by manf overexpression as well.
significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression.
gene name stands for the particular homologue gene name in drosophila.
notice that the same kegg identifier can lead to several different genes in drosophila.
the according coloured scheme for endocytosis kegg pathway is presented in figure  <dig>
to visualise manf expression at subcellular level we used 3rd instar larval salivary gland cells that are the largest ones found in drosophila.
in the basal part, there was partial colocalisation of manf expression with gfp-rab <dig>  as well as with early endosomal marker gfp-rab <dig> .
in larval fat body large cells with high secretory capacity gfp-clathrin light chain , a marker for clathrin coated vesicles, colocalised with manf in some structures .
manf localised close to gfp-rab <dig>  an important player in trafficking between the early and late endosomes and lysosomes, showing weak colocalisation .
thus manf localises to the endosomal structures with markers clc, rab <dig>  rab <dig>  and rab11; but probably does not share the same protein complexes with them.
ultrastructural analysis of manfmzΔ <dig> mutant stage  <dig> embryos revealed that the cells of secretory tissues such as gastric caeca, contain huge vesicles filled with cellular debris resembling multivesicular bodies and autophagosomes .
these structures were clearly missing in wild type embryos of the same age .
it is possible that these vesicles contain the misfolded proteins to be degraded or, alternatively, that the autophagy pathway is activated.
the accumulation of vesicles full of debris to be degraded could be also due to the blockage in endosomal trafficking or lysosomal degradation.
lysosomal genes are downregulated in manf mutants
because we detected in secretory cells the accumulation of multivesicular body like structures, is it possible that the lysosomal digestion mechanism was altered.
our microarray analysis revealed transcriptional change in 45% of lysosome related genes present in the kegg database.
many of them were downregulated in manfmzΔ <dig> embryos and some in manfΔ <dig> larvae .
the atpase v-type h+ transporting subunit that maintains acidic environment in lysosomes showed downregulation in both mutants but was upregulated in manf overexpressing larvae.
the expression of other lysosomal membrane proteins and several lysosomal hydrolases was also altered.
significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression.
id stands for the particular homologue gene name in drosophila.
notice that the same kegg identifier can lead to several different genes.
the according coloured scheme for lysosome kegg pathway is presented in additional file  <dig>
at the subcellular level, manf colocalises partially with er-targeted marker and very poorly if not at all with the lysosomal compartment .
nonetheless, it is possible that the lack of manf modifies the fusion of lysosomes with multivesicular body-like structures by some still unidentified mechanism.
paternal rescue of the manfmzΔ <dig> mutant embryos leads to reduction in the amount of differentially expressed genes
in drosophila, substantial bulk of transcribed mrnas and translated proteins necessary for early embryonic patterning and development are maternally contributed to the oocyte at high levels.
these mrnas and proteins are stored and used gradually during the embryogenesis and in some cases through the whole larval development.
when the embryonic lack of maternal manf was rescued by paternal wild type gene expression, the transcriptome changes were evident but restricted to a smaller number of genes than changes caused by the complete lack of manf.
as many as  <dig> genes were significantly upregulated by paternal rescue resulting in  <dig> functionally enriched go term clusters .
we obtained go terms related to response to stimulus and neurological process , consisting of genes like transcription factor pros, pumilio , pastrel , rop, rl, and small optic lobes .
transcripts of several genes coding membrane proteins also showed enrichment.
among them, we observed many genes coding ion-binding proteins  such as klumpfuss  and odd skipped .
other enriched go term clusters were membrane  together with cell division, cell cycle and cytoplasm .
only  <dig> genes were downregulated by paternal rescue giving  <dig> functional go term clusters .
ubiquitous overexpression of manf results in transcriptional activation and upregulation of genes involved in cell cycle and cell death
we used enhancer trap line 69b-gal <dig> to overexpress manf, which we obtained as a commonly known gal <dig> line with an epidermal expression pattern.
after careful mapping the expression pattern of 69b-gal <dig>  we detected broad gal <dig> expression in other tissues than epidermis - in central nervous system , imaginal discs , garland cells, ring gland, but neither in fat body nor in gastric caeca.
ectopic expression of manf under 69b-gal <dig> rescues completely manfΔ <dig> mutant lethality and the rescued adults are viable and fertile if maintained as a stock  <cit> .
when comparing the gene expression profiles between manf overexpressing and wild type larvae we found  <dig> genes upregulated that could be grouped in  <dig> functional go term clusters .
this gene set showed enrichment in processes related to regulation of gene expression, protein localisation and transport, and cell cycle .
genes involved in regulation of cell death were also upregulated .
according to the previous study in hela cells, knockdown of manf increased cell proliferation and susceptibility to er stress induced cell death  <cit> .
our results support the involvement of manf in regulation of cell cycle and cell death offering several candidate genes for further studies.
manf overexpression in larvae caused downregulation of  <dig> genes annotated in  <dig> functional clusters .
the most prominent group consisted of go terms such as membrane, plasma membrane, signal peptide, glycoprotein, disulfide bond, glycosylation site: n-linked , integral to membrane, and transmembrane .
the majority of processes related to these go terms take place in the er such as cleavage of the signal peptide and disulfide bond formation.
the main arthropod cuticular component chitin is composed of polymerised glcnac residues.
another prominent group was ion binding and metal binding .
axon guidance, cell projection organization, neuron development, axonal defasciculation, cell motion, cell recognition  were also enriched in line with our previous results implicating the role of manf in neuritogenesis  <cit> .
when comparing the upregulated genes in both paternally rescued embryos and in manf overexpressing larvae, the common represented go term clusters were ion binding , membrane fraction , oxidation reduction  and cell cycle  .
all together there were  <dig> annotated genes commonly upregulated by manf, among these well known genes like cbl, diaphanous , kinesin-like protein at 68d , rl, and rop.
among the downregulated genes in both paternal rescue and manf overexpression, there were only  <dig> genes in common e.g.
cg <dig>  a diacylglycerol kinase involved in phosphoinositol signalling and glycerolipid metabolism.
conclusively, in manf overexpression the typical growth factor signalling mediators rl and cbl were upregulated.
the links upstream of these mediators and downstream of secreted manf still remain missing.
the second cluster of genes was directly linked to membrane modifications and transport.
interestingly, the intracellular protein modification processes in er were also enhanced by manf overexpression.
so presumably manf has a dual role - one intracellularily in the er, and the other extracellularly after being secreted.
differentially regulated genes in larval manfΔ <dig> mutant in comparison to manf overexpression
next, we searched for genes showing downregulation in manfΔ <dig> larval mutant and were upregulated in manf overexpression condition, and vice versa.
altogether  <dig> probes on the microarray showed this opposite regulation resulting in  <dig> annotated drosophila genes .
in this group, there were genes responsible for locomotory behaviour , genes involved in oxidation reduction and metal binding , in cell division , genes coding membrane proteins , and genes involved in insulin signalling .
because of the diversity of go terms and low number of genes the functional annotation clustering did not give statistically significant results.
upr and parkinson's disease
among the known genes involved in pd,  <dig> are conserved between mammals and drosophila, and 44% of these were differentially expressed in our microarray assay .
importantly, several genes from dopamine uptake  and synthesis  were differentially expressed .
the expression of many genes involved in mitochondria and ubiquitin proteasome pathways were also altered.
increasing evidence indicates that organelle stress is a key event in neurodegeneration  <cit> .
upr is an adaptive process aiming to restore the cell homeostasis under er stress conditions and to re-establish the properly folded protein synthesis.
under irreversible er damage upr initiates cell death pathway to eliminate damaged cells.
manf could be responsible for the recovery and survival pathway in upr.
in this scenario, the lack of manf especially in the secretory cells with high rates of protein synthesis, including neurons with intensive neurotransmission, might result in shift from er stress to upr towards cell death.
however, the mechanism how manf dispatches this function is still unclear.
recently the structural homology between the manf c-terminal folding and sap domain of ku <dig> has been demonstrated  <cit> .
sap domain of ku <dig> is known to inhibit bax-induced apoptosis  <cit>  and in vitro experiments with mammalian cultured neurons showed that manf rescues the ngf-deprivation induced cell death as efficiently as ku <dig> itself  <cit> .
on the other hand we showed that the overexpression of manf resulted in upregulation of genes involved in oxidation reduction.
the da neurons are known to be very sensitive to oxidative stress because dopamine metabolites are highly oxidative compounds  <cit> .
therefore the upregulation of genes responsible for oxidation reduction could be already protective for da neurons.
besides reactive dopamine metabolites, the mitochondrial dysfunction has been implicated in the neurodegeneration occurring in pd.
we found that, in manfmzΔ <dig> mutants with degenerating da neurites, the nuclear genes coding for mitochondrial proteins were significantly enriched among the downregulated genes.
significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression.
notice that the same kegg identifier can lead to several different genes.
several drosophila genes associated with processes in pd missing from kegg pathway were added.
the according coloured scheme for parkinson's disease kegg pathway is presented in figure  <dig>
CONCLUSIONS
we have studied the gene expression changes of two slightly different manf mutants in drosophila.
surprisingly, the expression profiles of embryonic lethal manfmzΔ <dig> and larval lethal manfΔ <dig> mutants were quite diverse.
it might be due to the dissimilar roles of manf gene during embryonic and larval stages or it indicates the difference between the absolute lack of manf versus gradual diminishing of maternally contributed stores of manf gene products.
our microarray analysis followed by functional annotation clustering revealed statistically significant enrichment related to metabolism and membrane transport and transporters.
the observed changes in membrane traffic were supported by ultrastructural studies of manfmzΔ <dig> mutant.
more than 40% of known drosophila genes related to er and upr showed altered expression levels in manf mutants.
we found changed expression of several genes known to be associated with processes altered in pd such as oxidative phosphorylation, mitochondrial function, dopamine metabolism and uptake, and protein ubiquitinaton.
the lack of manf in manfmzΔ <dig> mutant embryos resulted in massive upregulation of stress, defense, and immune response related genes as well as genes involved in proteolysis and cell death.
overexpression of manf resulted in upregulation of genes involved in oxidation reduction, an important process to protect da neurons from oxidative stress.
thus, our results support the previously reported findings in mammalian cells that upregulation of manf is important in upr and could be protective for the cell.
it is also evident that er stress leads to upr and cell death in the absence of manf.
these effects were less drastic when manf was gradually abolished in manfΔ <dig> mutant larvae and related to several metabolic processes and downregulation of genes involved in replication, transcription and splicing.
still, stress and defense related genes were enriched among the upregulated genes of both manf mutants.

 recently characterised manf and cdnf form an independent family of neurotrophic factors. manf was identified from a conditioned media of cultured mesencephalic astrocytes in a search for secreted factors supporting dopamine neurons. specific loss of da neurons is the characteristic feature of parkinson's disease. therefore factors that maintain and support da neurons are attractive candidates for the treatment of pd. manf was shown to support the survival of cultured primary da neurons but to have no effect on cultured gabaergic or serotonergic neurons. subsequently, mammalian manf and its paralog cdnf were shown to prevent the loss of da neurons in mouse 6-ohda pd model. contrary to other vertebrate neurotrophic factors the manf/cdnf family is evolutionarily well conserved among multicellular organisms including the fruit fly, drosophila melanogaster. importantly, the protective role of manf for da neurons is also conserved. apparently both mammals and invertebrates share the same signalling partners as the lack of drosophila manf can be substituted by human manf. however, the interaction partners or how these proteins act at the molecular level are still elusive. it is important to understand the mechanisms of how these manf/cdnf family proteins work at molecular level before the potential therapeutic applications. recent studies have shown the protective role of mammalian manf to be more general than restricted to the nervous system. manf is upregulated by upr in several mammalian cell lines and by ischemia induced upr in the heart and brain. er is the central regulator of protein folding and quality control and it has to adapt its capacity to the specific need of a particular cell type. conditions challenging the function of the er, like an increase of newly synthesized unfolded proteins in its lumen, lead to upr. in eukaryotes, the three canonical branches of upr are mediated by er membrane-associated sensor proteins. in stress-free, functional er the intralumenal domains of these sensor proteins are bound to a chaperone bip/grp and maintained inactive. the upr intersect with a variety of inflammatory and stress signalling pathways and networks activated by oxidative stress, all of which can influence cell metabolism. er stress and upr have also been implicated in the pathogenesis of several neurodegenerative diseases because of their characteristic accumulation of specific misfolded proteins. data from pd patients reveal that in da neurons of substantia nigra the upr is activated. recently, drosophila has been used progressively to model human neurodegenerative diseases and upr. we have previously generated and characterized drosophila manf mutants. the zygotic null mutants survive up to 2nd instar larvae due to the high maternal contribution. mutants lacking both maternal and zygotic manf are late embryonic lethal and never hatch. the embryonic lack of maternal manf gene products and the lethality is rescued by paternal manf gene expression. both manfmzΔ and manfΔ mutants share nervous system defects, particularly dopamine neurites are altered and degenerate. ectopic overexpression of manf reveals no evident abnormalities in the embryonic or larval nervous system development or in the adult flies. here we compare the mrna expression profiles of manfmzΔ mutant embryos, manfΔ mutant larvae, paternally rescued maternal mutant embryos manfmΔ96/+, and manf ubiquitously overexpressing larvae to the wild type animals of exactly the same stages.  the most prevalent changes in gene expression occur in manf mutants that lack the maternal contribution of manf for microarray gene expression analysis we used two developmental stages in combination with three separate genotypes. the age of embryos and larvae were selected according to the lifespan of the manf mutants. manfmzΔ mutants fail in tracheal air filling and never hatch. mutant manfmzΔ embryos were picked during the late stage of just before hatching when the trachea of wild type embryos fill with air. mutant manfΔ larvae with maternal contribution survive to approximately hours ael and were collected as first instars 29- hours ael when maternal loads of manf gene products have decreased. three biologically independent sets of samples were used for microarray analysis. the expression profiles of all transgenic and mutant animals were compared to the wild type of the corresponding developmental stage. the numerical overview of statistically significant differences showed the most prominent changes in gene expression of manfmzΔ mutants. the smallest changes took place in the case of paternal rescue. among the differentially regulated genes, approximately half were up- or downregulated in different manf conditions. altogether we validated genes of the microarray results. genes were selected by several criteria such as differential expression or similar regulation in both mutants or otherwise high representation in the whole dataset. as a result, % of validated genes in different genetic conditions were independently confirmed by qpcr. the number of differentially regulated probes was compared. in the agilent drosophila microarray design there was unequal number of probes targeting the particular gene, ranging from one probe to several per gene. only statistically significant results are presented. results of qpcr were made comparable to microarray fold change by calculating the log from the relative fold changes. t-test means calculated p-value associated with student's t-test. only statistically significant results are presented. results of qpcr results were made comparable to microarray fold change by calculating the log of the relative fold changes. t-test means calculated p-value associated with student's t-test. membrane transporters and metabolic genes are downregulated in manfmzΔ mutants development of maternal and zygotic mutant manfmzΔ proceeds until stage with no differences to wild type embryos, but hours ael the cuticle and the nervous system defects become evident. in comparison to wild type embryos of the same age, in manfmzΔ mutants genes were found to be downregulated. these genes were grouped into functional clusters, among which the most significantly enriched clusters were related to membrane transporters and transmembrane proteins. there were several enriched clusters referring to different metabolic processes such as amine, amino acid and carboxylic acid catabolic processes, dna metabolic processes, and genes related to pyrimidine metabolism. clusters are represented starting from the highest enrichment score in diminishing order. only the highest clusters are shown, for the full list of david cluster analysis of downregulated genes in manfmzΔ mutants, see additional file in the case of missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above. among the downregulated genes in manfmzΔ mutants, the gene ontology term of mitochondria-related transcripts was highly enriched. mitochondria are the respiratory machinery of the cell responsible for oxidation processes and participate in maintaining cellular homeostasis. the lack of manf causes downregulation of several components in all mitochondrial compartments: the lumen as well as the inner and outer membranes. stress and defence response related genes are induced in manfmzΔ mutants in manfmzΔ mutant embryos, genes were upregulated in comparison to wild type embryos of the same stage. the most prominent group of significantly enriched go terms was immune and defense response, followed by the groups related to proteolysis, hydrolases and peptidases. the upregulated gene set was also enriched in genes related to actin cytoskeleton organization and actin filament-based process. moreover, genes involved in cell death were prominently enriched as well. clusters are represented starting from the highest enrichment score in diminishing order. only the highest clusters are shown, for the full list of david cluster analysis of upregulated genes in manfmzΔ mutants, see additional file in the case on missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above. enzymes for dopamine synthesis are upregulated despite of low dopamine levels extremely low dopamine levels are detected in manfmzΔ embryos. nonetheless, we observed significant upregulation of transcripts of the dopamine producing enzymes tyrosine hydroxylase and dopa decarboxylase. punch; encoding a gtp cyclohydrolase, an enzyme for tetrahydrobiopterin synthesis was also upregulated in manfmzΔ embryos. there could be several explanations for these alterations. tyrosine, the essential amino acid for dopamine synthesis, is transported into the cell from hemolymph. in manf mutants several amino acid membrane transporters were downregulated. the lack of substrate, tyrosine, together with low amounts of the end product, dopamine, could lead to the upregulation of the genes coding for the enzymes in dopamine synthesis pathway and their cofactors. genes involved in nucleic acid metabolism and protein folding are downregulated in larval manfΔ mutants larval manfΔ mutants with maternally contributed manf gene products never reach 3rd instar stage and rarely survive up to hours ael at 25°c. initially, manfΔ mutant larvae hatch and feed normally. whereas wild type larvae grow rapidly, the mutant larvae remain smaller and start to wander away from food, become sluggish and stop moving, still responding to touch and usually die during the first larval molt. because of the temporal differences in survival between individual manfΔ mutant larvae from 1st to 2nd instar, for microarray analysis we collected larvae 29- hours ael. when comparing the expression profile of larval manfΔ mutants to the wild type larvae, almost half the number of genes was significantly downregulated as compared to the rate in manfmzΔ embryos resulting in functional clusters. the most enriched go terms fell into clusters related to intracellular organelle lumen and nucleic acid metabolic processes. the cellular activities such as dna replication, rna processing and splicing were enriched among downregulated genes. the 5th highly enriched cluster consisted of go terms such as er related genes, proline and arginine metabolism, and oxioreductases. mitotic cell cycle, chromosomal segregation, and mitotic spindle organization were also clustered as significantly enriched. these changes could be linked to upr, as one of the outcomes of upr is general and unspecific downregulation of novel protein synthesis, at the same time activating the protein synthesis for chaperones and genes enhancing the protein folding to release the unfolded protein load in er. clusters are represented starting from the highest enrichment score in diminishing order. only the highest clusters are shown, for the full list of david cluster analysis of downregulated genes in manfΔ mutants, see additional file in the case on missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above. sugar metabolism, hydrolases, and er related oxidation reduction genes are induced in manfΔ larvae in manfΔ larval mutants, genes showed upregulation in comparison to the wild type larvae. the most enriched functional clusters included go terms like sugar metabolism and glucosidases, glycosyl hydrolases, and hydrolases and carboxylesterases, followed by cluster of monooxygenases, cytochrome p iron, vesicular fraction, oxidation reduction and endoplasmic reticulum. chitin and polysaccharide metabolism was also among the highly enriched go terms. the 5th ranked cluster of go terms was immune and defence response, which was the most highly enriched cluster in manfmzΔ mutant embryos. genes related to rna metabolism, atp binding, and dna replication are downregulated in both manf mutants next, we looked for functional terms among the commonly downregulated genes in both manf mutants. there was 30% of overlap in gene sets between the manfΔ and manfmzΔ mutants. among the downregulated overlapping genes, the enrichment of go terms fell into rna metabolism and ribosome biogenesis. around 10% of all known atp binding genes were downregulated together with genes of the purine and pyrimidine metabolism. additionally, the genes coding sugar transporters and the genes involved in transmembrane transport highly represented among downregulated genes in manfmzΔ mutant embryos, were repressed in zygotic mutant manfΔ as well. go terms related to dna replication were also enriched among the commonly downregulated genes in both manf mutants. altogether, the manfmzΔ mutant embryos lacking both maternal and zygotic manf showed twice more drastic decline from wild type transcriptome than manfΔ larval mutants, whose maternal transcripts gradually diminish. beside behavioural and growth phenotype, manfΔ larvae show degeneration of dopaminergic neurites in ventral nerve cord. we found three genes downregulated in both mutants that are involved in neurite development: abelson tyrosine kinase, guanine nucleotide exchange factor gef64c and the transcription factor longitudinals lacking. a third of all upregulated genes were induced in both mutants. immune and defense response was the most enriched functional cluster along with the group consisting of monooxygenases, oxidoreductases, vesicular fraction, endoplasmic reticulum, cytochrome p and lipid metabolic process. controversially, disabled, was upregulated among the genes involved in neuronal development e.g. transcription factor krüppel, negative regulator of growth shrub, insulin receptor, and drosophila extracellular-signal-regulated kinase rolled. genes related to upr were upregulated in manf mutants previous in vitro studies using tunicamycin, the inhibitor of glycosylation, to induce er stress in mammalian cell lines have shown in upr the upregulation of manf. in rat neonatal cardiomyocytes in response to upr manf is secreted to promote cellular survival. er stress and one of the consequences, upr, has been mainly studied in yeast and mammalian cells. in drosophila, there are several recent studies where upr has been addressed. manf has been shown to be upregulated after feeding tunicamycin to adult fruit flies indicating the involvement of manf in chemically induced upr in drosophila. to find out the intracellular localisation of manf in drosophila, we used larval 2nd instar garland cells. garland cells are nephrocytes with high rate of endocytosis and express several neuronal and exocytosis markers e.g. prospero, snare binding protein ras opposite facilitating neurotransmitter secretion, and syntaxin 1a. these cells have the most abundant expression of manf starting from embryogenesis. in the garland cells, manf was localised around the nucleus, partially overlapping with er-targeted marker. next, we tested the hypothesis that the metabolic changes in manf mutant could be the result of severe er stress caused by altered expression of er related genes. drosophila genes homologous to several er stress pathway have been identified. out of genes involved in er and protein processing in the kegg database, have one or more homologues in fruit flies. of these upr related drosophila genes, 30% showed altered gene expression in our microarray experiment. altogether, genes involved in er and protein processing show statistically significant expression changes. the gene cg is an annotated gene with unknown function in drosophila. its human homologue nucleotide exchange factor sil is a bip binding protein. in humans, several mutations in sil gene disrupting the protein cause the marinesco-sjögren syndrome, an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy. we validated cg by qpcr as downregulated by manf overexpression and upregulated when manf is abolished in drosophila embryos and larvae. it has been shown by immunoprecipitation studies that mammalian manf binds to bip. thus it is possible that manf and cg compete in binding to bip together with unfolded proteins. as the ectopic overexpression of manf has no effect on fruit fly viability or nervous system development, the diminished transcript level for cg is not comparable to the total lack of this gene product in the mss patients. according to our qpcr validated microarray results several other genes implicated in upr were downregulated in larvae overexpressing manf, such as pancreatic eif-2α kinase, heat shock protein, ubiquilin, and septin interacting protein. in embryonic manfmzΔ mutants all above mentioned genes were significantly upregulated as well as considerable number of other er chaperone genes. furthermore, when evaluating the ultrastructural changes in manfmzΔ mutants, we noticed that the er was swollen and dilated in epidermal cells, indicating severe disturbances of er structure. in manfmzΔ mutant embryos the extent of phosphorylated eukaryotic initiation factor eif2α was more than two fold upregulated when compared to the wild type indicating the presence of upr in these manf mutants. the phosphorylation of eif2α by perk is a hallmark for upr, resulting in reversible blockage of translation and downregulation of the protein load to the er. in drosophila there are two kinases, perk and gcn shown to be able to phosphorylate eif2α. the expression of gcn is high only during early stages of embryogenesis. thus perk is a potential candidate kinase behind eif2α phosphorylation at the end of embryogenesis. interestingly, our microarray data showed that in manfmzΔ mutants the transcription of perk was upregulated and the genes involved in different metabolic processes such as amino acid, dna and pyrimidine metabolism were downregulated indicating overall inhibition of translation. so it is probable that the upr perk pathway is activated in manfmzΔ mutants. the second upr sensor, ire activates two separate downstream branches. one of the branches leads to the activation of jun kinase and death pathway. according to the microarray results, drosophila jun kinase kinase hemipterous showed significant upregulation in both manf mutants. significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. gene name stands for the particular homologue gene name in drosophila. notice that the same kegg identifier can lead to several different genes. p.res = paternal rescue, mut = mutant, o.ex. = overexpression. conclusively, the absolute lack of manf results in severe er stress and upregulation of many genes involved in upr finally leading to the cell death. when maternal manf stores are gradually decreased in manfΔ larvae, there are only few genes upregulated that are related to er: cg ubiquitin protein ligase lin heat shock protein cognate and ubiquitin-conjugating enzyme cg as our data come mostly from gene expression analyses, further biochemical experiments are needed to identify the exact role of manf in upr. lack of manf results in downregulation of several genes in exocytosis pathway ultrastructural study of manfmzΔ mutants revealed overload of vesicles next to the apical part of epidermal cells and reduced microvillae thought to enhance the capacity of the secretion of these cells. this result, together with the severe defects observed in the cuticle secretion and organisation, suggested a possible involvement of the genes of the exocytosis pathway. indeed, the expression of several genes related to exocytosis and snare transport were altered in different manf conditions. in manf mutants, several genes implied in exocytosis and vesicle transport from golgi complex to the plasma membrane were downregulated, whereas the er residing syntaxins - stx and stx - were upregulated. this supports an inhibition of secretion from golgi complex to the plasma membrane as seen in manfmzΔ mutant epidermal cells in vesicle accumulation close to the apical area. significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. gene name stands for the particular homologue gene name in drosophila. few related drosophila genes associated with neurotransmission missing from kegg pathway were added. the according coloured scheme for exocytosis and snare of kegg pathway is presented in additional file p.res = paternal rescue, mut = mutant, o.ex. = overexpression. expression of genes involved in cuticle development were altered in manfmzΔ mutants we have previously shown that manfmzΔ embryos have disorganized cuticle. at the end of embryogenesis from stage onward, the cuticle components are secreted by epithelial cells and stored in regular layers, and subsequently the cuticular proteins are crosslinked by dopamine-derived quinones. among the downregulated genes in manfmzΔ embryos, there were genes coding the structural components of the insect cuticle. at the same time, several other genes responsible for cuticle development were upregulated, such as the genes encoding enzymes involved in chitin synthesis, krotzkopf verkehrt, knickkopf, and syx1a. additionally, several genes involved in epithelial development and morphogenesis were upregulated and significantly enriched among the go terms. we used transmission electron microscopy analysis in manfmzΔ mutants at the embryonic stage to investigate the epithelial cells responsible for cuticle secretion. indeed, these cells showed morphologically abnormal er and accumulation of vesicles in the apical part. it is possible that the enhanced endocytosis and disturbed exocytosis, together with misbalance in cuticular components, lead to disorganised and disrupted cuticle in manfmzΔ mutant embryos. in larval manfΔ mutant with gradually fading maternal contribution, the cuticle showed no disruption and the chitin layers were deposited and organised normally. instead there were problems in shedding the old cuticle and often the 1st instar cuticle remained attached. this implies that the maternal loading of manf gene products in larval manfΔ mutants was sufficient to overcome defects in early cuticle development, secretion and layering, but insufficient to complete the first molt. large vesicles filled with electron dense debris are accumulated in manfmzΔ mutant to investigate the routes of membrane trafficking we evaluated genes involved in endocytosis. of all drosophila homologues known to be involved in endocytosis, 47% showed significant expression changes in our microarray experiment. genes coding for components of multivesicular body formation were especially altered. several transmembrane receptors of growth factors were downregulated in manf mutants and upregulated when manf was overexpressed. pdgf- and vegf-receptor related pvr was upregulated in larvae in both lack and overexpression of manf. cbl, an e ubiquitin ligase and negative regulator of tyrosine kinase receptor signalling, was downregulated in mutant larvae and upregulated under manf overexpression conditions. two different members of endosomal recycling pathway, par family members and rab-protein were upregulated in mutants. par transcripts were upregulated by manf overexpression as well. significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. gene name stands for the particular homologue gene name in drosophila. notice that the same kegg identifier can lead to several different genes in drosophila. the according coloured scheme for endocytosis kegg pathway is presented in figure to visualise manf expression at subcellular level we used 3rd instar larval salivary gland cells that are the largest ones found in drosophila. in the basal part, there was partial colocalisation of manf expression with gfp-rab as well as with early endosomal marker gfp-rab. in larval fat body large cells with high secretory capacity gfp-clathrin light chain, a marker for clathrin coated vesicles, colocalised with manf in some structures. manf localised close to gfp-rab an important player in trafficking between the early and late endosomes and lysosomes, showing weak colocalisation. thus manf localises to the endosomal structures with markers clc, rab rab and rab11; but probably does not share the same protein complexes with them. ultrastructural analysis of manfmzΔ mutant stage embryos revealed that the cells of secretory tissues such as gastric caeca, contain huge vesicles filled with cellular debris resembling multivesicular bodies and autophagosomes. these structures were clearly missing in wild type embryos of the same age. it is possible that these vesicles contain the misfolded proteins to be degraded or, alternatively, that the autophagy pathway is activated. the accumulation of vesicles full of debris to be degraded could be also due to the blockage in endosomal trafficking or lysosomal degradation. lysosomal genes are downregulated in manf mutants because we detected in secretory cells the accumulation of multivesicular body like structures, is it possible that the lysosomal digestion mechanism was altered. our microarray analysis revealed transcriptional change in 45% of lysosome related genes present in the kegg database. many of them were downregulated in manfmzΔ embryos and some in manfΔ larvae. the atpase v-type h+ transporting subunit that maintains acidic environment in lysosomes showed downregulation in both mutants but was upregulated in manf overexpressing larvae. the expression of other lysosomal membrane proteins and several lysosomal hydrolases was also altered. significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. id stands for the particular homologue gene name in drosophila. notice that the same kegg identifier can lead to several different genes. the according coloured scheme for lysosome kegg pathway is presented in additional file at the subcellular level, manf colocalises partially with er-targeted marker and very poorly if not at all with the lysosomal compartment. nonetheless, it is possible that the lack of manf modifies the fusion of lysosomes with multivesicular body-like structures by some still unidentified mechanism. paternal rescue of the manfmzΔ mutant embryos leads to reduction in the amount of differentially expressed genes in drosophila, substantial bulk of transcribed mrnas and translated proteins necessary for early embryonic patterning and development are maternally contributed to the oocyte at high levels. these mrnas and proteins are stored and used gradually during the embryogenesis and in some cases through the whole larval development. when the embryonic lack of maternal manf was rescued by paternal wild type gene expression, the transcriptome changes were evident but restricted to a smaller number of genes than changes caused by the complete lack of manf. as many as genes were significantly upregulated by paternal rescue resulting in functionally enriched go term clusters. we obtained go terms related to response to stimulus and neurological process, consisting of genes like transcription factor pros, pumilio, pastrel, rop, rl, and small optic lobes. transcripts of several genes coding membrane proteins also showed enrichment. among them, we observed many genes coding ion-binding proteins such as klumpfuss and odd skipped. other enriched go term clusters were membrane together with cell division, cell cycle and cytoplasm. only genes were downregulated by paternal rescue giving functional go term clusters. ubiquitous overexpression of manf results in transcriptional activation and upregulation of genes involved in cell cycle and cell death we used enhancer trap line 69b-gal to overexpress manf, which we obtained as a commonly known gal line with an epidermal expression pattern. after careful mapping the expression pattern of 69b-gal we detected broad gal expression in other tissues than epidermis - in central nervous system, imaginal discs, garland cells, ring gland, but neither in fat body nor in gastric caeca. ectopic expression of manf under 69b-gal rescues completely manfΔ mutant lethality and the rescued adults are viable and fertile if maintained as a stock. when comparing the gene expression profiles between manf overexpressing and wild type larvae we found genes upregulated that could be grouped in functional go term clusters. this gene set showed enrichment in processes related to regulation of gene expression, protein localisation and transport, and cell cycle. genes involved in regulation of cell death were also upregulated. according to the previous study in hela cells, knockdown of manf increased cell proliferation and susceptibility to er stress induced cell death. our results support the involvement of manf in regulation of cell cycle and cell death offering several candidate genes for further studies. manf overexpression in larvae caused downregulation of genes annotated in functional clusters. the most prominent group consisted of go terms such as membrane, plasma membrane, signal peptide, glycoprotein, disulfide bond, glycosylation site: n-linked, integral to membrane, and transmembrane. the majority of processes related to these go terms take place in the er such as cleavage of the signal peptide and disulfide bond formation. the main arthropod cuticular component chitin is composed of polymerised glcnac residues. another prominent group was ion binding and metal binding. axon guidance, cell projection organization, neuron development, axonal defasciculation, cell motion, cell recognition were also enriched in line with our previous results implicating the role of manf in neuritogenesis. when comparing the upregulated genes in both paternally rescued embryos and in manf overexpressing larvae, the common represented go term clusters were ion binding, membrane fraction, oxidation reduction and cell cycle. all together there were annotated genes commonly upregulated by manf, among these well known genes like cbl, diaphanous, kinesin-like protein at 68d, rl, and rop. among the downregulated genes in both paternal rescue and manf overexpression, there were only genes in common e.g. cg a diacylglycerol kinase involved in phosphoinositol signalling and glycerolipid metabolism. conclusively, in manf overexpression the typical growth factor signalling mediators rl and cbl were upregulated. the links upstream of these mediators and downstream of secreted manf still remain missing. the second cluster of genes was directly linked to membrane modifications and transport. interestingly, the intracellular protein modification processes in er were also enhanced by manf overexpression. so presumably manf has a dual role - one intracellularily in the er, and the other extracellularly after being secreted. differentially regulated genes in larval manfΔ mutant in comparison to manf overexpression next, we searched for genes showing downregulation in manfΔ larval mutant and were upregulated in manf overexpression condition, and vice versa. altogether probes on the microarray showed this opposite regulation resulting in annotated drosophila genes. in this group, there were genes responsible for locomotory behaviour, genes involved in oxidation reduction and metal binding, in cell division, genes coding membrane proteins, and genes involved in insulin signalling. because of the diversity of go terms and low number of genes the functional annotation clustering did not give statistically significant results. upr and parkinson's disease among the known genes involved in pd, are conserved between mammals and drosophila, and 44% of these were differentially expressed in our microarray assay. importantly, several genes from dopamine uptake and synthesis were differentially expressed. the expression of many genes involved in mitochondria and ubiquitin proteasome pathways were also altered. increasing evidence indicates that organelle stress is a key event in neurodegeneration. upr is an adaptive process aiming to restore the cell homeostasis under er stress conditions and to re-establish the properly folded protein synthesis. under irreversible er damage upr initiates cell death pathway to eliminate damaged cells. manf could be responsible for the recovery and survival pathway in upr. in this scenario, the lack of manf especially in the secretory cells with high rates of protein synthesis, including neurons with intensive neurotransmission, might result in shift from er stress to upr towards cell death. however, the mechanism how manf dispatches this function is still unclear. recently the structural homology between the manf c-terminal folding and sap domain of ku has been demonstrated. sap domain of ku is known to inhibit bax-induced apoptosis and in vitro experiments with mammalian cultured neurons showed that manf rescues the ngf-deprivation induced cell death as efficiently as ku itself. on the other hand we showed that the overexpression of manf resulted in upregulation of genes involved in oxidation reduction. the da neurons are known to be very sensitive to oxidative stress because dopamine metabolites are highly oxidative compounds. therefore the upregulation of genes responsible for oxidation reduction could be already protective for da neurons. besides reactive dopamine metabolites, the mitochondrial dysfunction has been implicated in the neurodegeneration occurring in pd. we found that, in manfmzΔ mutants with degenerating da neurites, the nuclear genes coding for mitochondrial proteins were significantly enriched among the downregulated genes. significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. notice that the same kegg identifier can lead to several different genes. several drosophila genes associated with processes in pd missing from kegg pathway were added. the according coloured scheme for parkinson's disease kegg pathway is presented in figure  we have studied the gene expression changes of two slightly different manf mutants in drosophila. surprisingly, the expression profiles of embryonic lethal manfmzΔ and larval lethal manfΔ mutants were quite diverse. it might be due to the dissimilar roles of manf gene during embryonic and larval stages or it indicates the difference between the absolute lack of manf versus gradual diminishing of maternally contributed stores of manf gene products. our microarray analysis followed by functional annotation clustering revealed statistically significant enrichment related to metabolism and membrane transport and transporters. the observed changes in membrane traffic were supported by ultrastructural studies of manfmzΔ mutant. more than 40% of known drosophila genes related to er and upr showed altered expression levels in manf mutants. we found changed expression of several genes known to be associated with processes altered in pd such as oxidative phosphorylation, mitochondrial function, dopamine metabolism and uptake, and protein ubiquitinaton. the lack of manf in manfmzΔ mutant embryos resulted in massive upregulation of stress, defense, and immune response related genes as well as genes involved in proteolysis and cell death. overexpression of manf resulted in upregulation of genes involved in oxidation reduction, an important process to protect da neurons from oxidative stress. thus, our results support the previously reported findings in mammalian cells that upregulation of manf is important in upr and could be protective for the cell. it is also evident that er stress leads to upr and cell death in the absence of manf. these effects were less drastic when manf was gradually abolished in manfΔ mutant larvae and related to several metabolic processes and downregulation of genes involved in replication, transcription and splicing. still, stress and defense related genes were enriched among the upregulated genes of both manf mutants. 
when evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic manf mutants, the endoplasmic reticulum stress and membrane traffic alterations were evident. in drosophila manf mutants the expression of several genes involved in parkinson's disease was altered as well. the most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. in the absence of manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. in neurodegenerative diseases, such as pd, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. to enlighten the role of manf in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of drosophila manf.
 manf and cdnf are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. to date, the receptors and signalling pathways of this novel manf/cdnf family have remained unknown. independent studies have showed upregulation of manf by unfolded protein response. to enlighten the role of manf in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of drosophila manf.  the most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. when evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic manf mutants, the endoplasmic reticulum stress and membrane traffic alterations were evident. in drosophila manf mutants the expression of several genes involved in parkinson's disease was altered as well.  we conclude that besides a neurotrophic factor, manf is an important cellular survival factor needed to overcome the upr especially in tissues with high secretory function. in the absence of manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. in neurodegenerative diseases, such as pd, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. the degeneration of dopaminergic neurons is the hallmark for pd and our work provides a clue on the mechanisms by which the novel neurotrophic factor manf protects these neurons. 