BACKGROUND
membrane-associated guanylate kinases  form a family of scaffolding proteins, engaged in the organisation of multiprotein complexes, which are often associated with cellular junctions or signalling complexes, such as the vertebrate tight junction  or the drosophila septate junction  in epithelial cells or the neuromuscular junction .
their capacity to serve as a platform for recruiting larger protein assemblies results from the presence of several protein-protein interaction domains: one to three pdz -1)-domains, an sh3--domain and a guanylate kinase -domain.
some members additionally contain one or two l <dig> -domains in their n-terminus  <cit> .
this modular structure is ideally suited to recruit a variety of components into a protein complex, the composition of which often depends on the cell type and/or developmental stage.
in addition, maguk-encoding genes often give rise to more than one isoform by alternative splicing, thus increasing the possibility of multiple interactions, localisations and/or functions.
for example, the vertebrate membrane-associated palmitoylated protein  <dig>  encodes a retina-specific isoform, which lacks the l <dig> domain in its n-terminus  <cit> .
similarly, drosophila discs large  expresses an isoform  lacking the l <dig> domain, which is unable to bind to dlin- <dig> in epithelia, whereas an isoform containing the l <dig> domain  can associate with dlin- <dig> in the neuromuscular junction  <cit> .
drosophila polychaetoid, the orthologue of mammalian zo- <dig>  encodes two isoforms, one of which is localised apically, whereas the other one distributes more broadly along the lateral membrane of epithelial cells of the wing imaginal disc  <cit> .
finally, drosophila stardust  expresses a distinct isoform in the embryo, characterised by the presence of a particular exon, which is not expressed in the eye  <cit> .
taken together, maguk proteins provide a versatile platform for the dynamic assembly of protein complexes in various cell types.
septate junctions  in epithelia of drosophila reside in the lateral membrane, basal to the zonula adherens .
two kinds of sjs are known, pleated sjs and smooth sjs.
pleated sjs are present in most ectodermally derived epithelia of the embryo, i. e. the epidermis, the tracheae, the salivary glands, the fore- and the hindgut, but they are excluded from the amnioserosa and the malpighian tubules.
they are characterised by ladder-like septa spanning the space between the lateral membranes of adjacent cells.
they are first visible in these epithelia around stage  <dig> and become further elaborated by the end of embryogenesis  <cit> .
sjs can be found in epithelia of arthropods  <cit>  and at the blood-brain barrier of both arthropods and chordates  <cit>  .
they serve as barriers between different compartments by providing a paracellular diffusion barrier, a function equivalent to that of vertebrate tight junctions.
in addition, sjs are involved in the control of tracheal tube size  <cit> .
the molecular components in drosophila septate junctions include transmembrane proteins, such as neurexin iv   <cit> , neuroglian, gliotactin and a na+/k+ atpase  <cit> , the cell surface protein lachesin  <cit>  as well as scaffolding proteins, such as the ferm  family member coracle   <cit>  and the maguk discs large   <cit> .
some of them are orthologues to vertebrate tj proteins, such as megatrachea or sinuous, two claudin-like proteins  <cit> , or contactin  <cit>  .
recently it has been shown that septate junctions contain an additional member of the maguk protein family, encoded by varicose, which is required for proper localisation of other septate junction components and tracheal tube size development  <cit> .
here we show by electron microscopy that varicose mutant embryos fail to develop proper pleated sjs.
we further demonstrate that two different transcripts and proteins are expressed in the embryo, which are differentially affected by different varicose alleles, pointing to individual functions.
in addition, we provide data to show that varicose is expressed in the wing and eye imaginal discs and necessary for their normal development.
RESULTS
varicose expresses two maguk isoforms in the embryo
in drosophila epithelia, the maguk-proteins stardust  and discs large  are required to organise protein complexes in the subapical and the lateral membrane, respectively.
sdt recruits the transmembrane protein crumbs  and two other scaffolding proteins, dlin- <dig> and dpatj, into a complex, which is localised subapically, i. e. in a small region of the apical plasma membrane just apical to the zonula adherens, in epithelial and photoreceptor cells .
we were interested in identifying additional partners of the crumbs complex, which might have a function in the organisation of epithelia of the drosophila embryo.
therefore, we took advantage of the recently published protein interaction map  <cit>  to screen for partners of dlin- <dig>  a component of the crumbs complex  <cit> .
one of the putative partners identified in this screen is encoded by drosophila cg <dig>  located at 38e <dig> on the left arm of the second chromosome.
it is predicted to encode three isoforms, which differ in their size and are the result of differential transcriptional initiation and alternative splicing  .
as demonstrated recently  <cit> , these proteins are encoded by varicose  and define a new subgroup of maguk proteins, which includes the mammalian proteins mpp <dig> and mpp6/vam-1/pals <dig> .
previously, only a single l <dig> domain was predicted in the longer isoform.
when using smart and reciprocal blasts , two l <dig> domains are predicted.
this is in agreement with the domain structure of orthologues from the honeybee, mouse, xenopus and zebrafish .
strikingly, human mpp <dig> also encodes two isoforms of different size, mpp6_a and mpp6_c, the longer of which carries an extended n-terminal region with two l <dig> domains.
the expression of two different transcripts in the embryo was verified by northern blots and rt-pcr.
using a probe common to all predicted transcripts, two mrnas of about  <dig>  and  <dig>  kb were detected on northern blots with poly-rna of staged wild-type embryos, with the shorter one being much more abundant than the longer one .
the expression of the two different transcripts was confirmed by rt-pcr, using transcript-specific primers .
however, rt-pcr experiments failed to detect vari transcripts encoding the predicted isoform d , suggesting that only isoforms b and c are expressed in the developing embryo, which represent the short and the long vari isoform, respectively.
in-situ hybridisations with a probe common to both transcripts reveal that vari is expressed in most ectodermally derived embryonic epithelia, i. e. the epidermis, the fore- and the hindgut, the tracheae and the salivary glands, but not in the malpighian tubules .
western blot analysis with antibodies raised against a recombinant protein containing the pdz- and the sh3-domain  revealed that two variants of ~ <dig> and ~ <dig> kda are present in extracts of wild-type or vari-hemizygous embryos .
the smaller isoform, which matches the predicted size , was already detectable in extracts from 0– <dig> hour embryos, but was much more abundant during later stages of embryogenesis.
the larger isoform  was only detectable in embryos older than  <dig> hours, and present in only very small amounts compared to the shorter form.
western blots from embryos expressing a flag-tagged vari-long protein confirmed the specificity of the larger isoform .
both proteins were almost completely or completely absent in extracts of embryos expressing vari-rnai or embryos homozygous for a deficiency, which removes the entire gene, respectively .
similar as shown recently  <cit> , our antibody recognised an epitope in most ectodermally derived epithelia of wild-type embryos, i. e. in the epidermis, the tracheae, the salivary glands, the fore- and the hindgut, but not in the malpighian tubules .
in these tissues, the protein detected by the antibody localised basal to the za, as revealed by double-staining with anti-crumbs  and anti-armadillo , respectively .
vari co-localised with the septate junction components neurexin iv  and discs large  , confirming its association with septate junctions.
varicose is required for septate junction development
we induced a vari mutation by imprecise excision of a p-element of the line pd <dig> , which localised immediately 5' to the transcription start site of the smaller transcript .
one out of  <dig> homozygous lethal excision lines isolated failed to complement deficiency dfds <dig> and vari alleles vari03953b, vari <dig>  varir <dig>  varir <dig> and varif <dig>  <cit> .
the mutant excision line carries a deletion that removes  <dig>  basepairs of genomic dna, thus completely eliminating exon 3a, the first exon of the transcript encoding the shorter isoform  plus part of the following intron .
consistent with this result, embryos homozygous mutant for this small deletion did not express any vari-short protein .
the genetic and molecular data classified the mutation identified here as a vari allele, which is called varimd <dig> from here on.
embryos homozygous mutant for varimd <dig> or transheterozygous for varimd109/vari03953b died at the end of embryogenesis with convoluted tracheal tubes , a phenotype similar to that described for vari03953b  <cit> .
apical-basal polarity was not affected, as revealed by proper apical localisation of crb .
the same phenotype was achieved by specifically knocking down vari function in the tracheae by expressing vari-rnai by means of btlgal <dig> .
septate junction components, such as coracle or neurexin iv, which are restricted to the lateral membrane in wild-type embryos, became distributed to all membranes in vari mutant embryos, both in the tracheae  and the epidermis .
this phenotype was reminiscent to that achieved in the absence of neurexin iv function .
based on the failure to properly localise the septate-junction-associated proteins neurexin iv and coracle  in vari mutant embryos  wu et al.
suggested that these embryos fail to properly establish and/or maintain the septate junctions.
in order to prove this, we carried out electron microscopic analysis of wild-type  and vari mutant embryos.
in wild-type embryos, pleated septate junctions are clearly distinguishable from stage  <dig> onwards  <cit>  by the presence of septa, which span the space between the lateral membranes of neighbouring cells, both in the epidermis  and the tracheae .
homozygous mutant varimd <dig> embryos of the same stage lacked septate junctions in these tissues .
similarly, septate junctions were not detected in vari03953b homozygous mutant embryos .
the za was not affected in epithelia of the mutant embryos and still formed a continuous apical belt.
this result finally proves that vari has an essential function in the formation of the pleated septate junctions during embryonic development.
varicose alleles differentially affect the two protein isoforms in the embryo
the molecular lesions in the dna of several vari alleles, which exhibit a weak, an intermediate or a strong mutant phenotype, respectively, have been described  <cit> .
in none of these mutant embryos, nor in embryos mutant for varimd <dig>  any localised vari protein could be detected in the embryo   <cit> .
we were interested to find out how the different alleles affect the expression of the two vari isoforms and whether there is any relationship between the strength of the mutant phenotype of a given allele as determined by genetic analyses and the expression of the two vari isoforms.
therefore, we performed rt-pcr and western blots on extracts of homozygous mutant embryos .
as expected, embryos mutant for the intermediate allele varimd <dig>  in which the transcription start site of the smaller form is deleted , did not express vari-short rna or vari-short protein, but still exhibited vari-long protein expression, which seemed even slightly up-regulated.
in the intermediate allele vari03953b, which carries a  <dig> bp deletion in the third common intron, very low amounts of vari-short rna and protein were expressed.
although vari-long transcripts were upregulated in the mutant embryos, the amount of vari-long protein was reduced in comparison to wild-type.
the null allele varir <dig> carries a point mutation leading to a premature stop codon in the region encoding the hook domain, which should result in a truncation of both protein isoforms.
as expected, this mutation did not affect the size of the two transcripts.
western blots revealed that the long isoform was in fact truncated: a protein of about  <dig> kd, corresponding to a truncated vari-long  could be detected.
the absence of any truncated short isoform suggested that this truncated form was not stable.
the null allele varif <dig>  which carries a transposon in the intron between exons 3a and  <dig>  did not express vari-long and only very low amounts of vari-short protein.
in contrast, the insertion line pd <dig>  which carries a p-element in the intron between exon  <dig> and 3a  expressed normal amounts of both proteins .
the mutation in vari <dig>  also characterised as a null allele, alters the splice acceptor site of the common exon  <dig>
therefore, one would expect that exon  <dig> is skipped.
as a consequence, both transcripts should be reduced by  <dig> nucleotides and both proteins should lack  <dig> amino acids.
surprisingly, vari <dig> mutant embryos still expressed wild-type sized transcripts and proteins.
however, sequencing the rt-pcr product obtained by amplification with primers vari-short- <dig> and vari-short- <dig>  revealed, that the mutant splice site is skipped, and the next ag-site inside exon  <dig> is used as a splice acceptor instead.
this results in a  <dig> bp deletion, which exactly removes  <dig> amino acids  of the sh3-domain, without affecting the reading frame.
this deletion is too small to be detected in the length of the rt-pcr product or the protein.
in the strong allele varir <dig>  which carries a single point mutation, resulting in the exchange of an arginine to a cysteine in the hook domain, the mutation did not have any effect on the size of the two proteins, but vari-long seemed to be present in lower amount.
in the weak allele vari38efa <dig>  which carries a mutation in the splice acceptor site of exon  <dig>  the rnas of both isoforms were reduced in length, but, as expected, only the long protein isoform was truncated.
taken together, the results obtained by western blot analysis showed that in most vari mutant alleles at least one of the protein isoforms is affected.
varicose is necessary, but not sufficient for neurexin iv localisation
the c-terminus of neurexin iv was able to pull-down the recombinant, his-tagged pdz-domain of varicose when fused to gst   <cit> .
we could further identify the c-terminal pdz-binding motif-eifi of neurexin iv as being crucial for this interaction, since its removal  completely abolished the interaction .
to confirm that these two proteins also form a complex in vivo, we made use of a protein trap line expressing a neurexin iv-gfp fusion protein, in which the p-element is inserted in the first intron and gfp is fused in frame right after the signal sequence .
immunoprecipitation of neurexin iv-gfp with an anti-gfp antibody from embryonic extracts co-precipitated both varicose isoforms .
it has recently been shown that neurexin iv is necessary to recruit varicose to the septate junctions  <cit> .
to demonstrate that it is also sufficient, we overexpressed neurexin iv in the posterior compartment of each segment, making use of an ep-insertion in the neurexin iv locus.
this induced a moderate enrichment of vari protein in regions of the cell with higher nrxiv protein levels .
overexpression of another sj component, dlga, failed to recruit vari to ectopic sites , further demonstrating the specificity of the nrxiv-vari interaction.
in contrast, vari-long did not recruit more nrxiv to the membrane , suggesting that vari is necessary, but not sufficient for nrxiv localisation.
varicose has initially been described as an interaction partner of dlin- <dig> in a yeast two-hybrid screen  <cit> .
this interaction could be confirmed by pull-down experiments.
a gst-fusion protein, containing the l27-domain of dlin- <dig>  pulled down the recombinant l <dig> domains of vari-long, fused to a his-tag.
the pdz-domain of dlin- <dig> was unable to mediate this interaction .
however, unlike nrxiv, overexpression of dlin- <dig> in the embryonic epidermis had no effect on the distribution of vari .
varicose is required for normal wing and eye development
the data show that varicose has an essential function during development of the embryo.
vari protein is also expressed at later stages, in the wing and the eye imaginal discs.
in the wing imaginal disc, vari is expressed throughout the disc epithelium, where it localises basal to crumbs, which marks the apical pole .
in the eye disc of third instar larvae, there is enriched expression of vari behind the morphogenetic furrow .
nrxiv and coracle, two other components of the septate junction, exhibit the same expression pattern   <cit> , suggesting that vari is associated with sj in differentiating ommatidia .
in both the eye and the wing discs, both isoforms could be detected, while ovaries expressed only vari-short, but not vari-long .
to assess vari function in imaginal discs, we expressed double-stranded rna  in a tissue-specific manner.
overexpression of vari-rnai by means of ptcgal <dig>  i. e. in a stripe of cells along the anterior-posterior compartment boundary of the wing imaginal disc, almost completely removed vari protein from these cells , suggesting that vari function was strongly suppressed.
reduction of vari had no effect on apical-basal polarity as revealed by the continuous apical localisation of crumbs .
use of sdgal <dig> to express vari-rnai in the developing wing pouch from first/second larval instar onwards gave rise to adult flies with defective wings or pharate adults, depending on the strength of the different vari-rnai effector-strains used.
hatched flies exhibited malformations of their wings to various degrees.
most prominent were gaps in the wing margin and defects in the unfolding of the wing .
eygal4>uas vari-rnai animals, which expressed the vari dsrna under the control of the eyeless promotor, i. e. in the eye imaginal discs from early stages onward as well as in a small stripe in the antennal disc  <cit> , developed defects in the adult eye.
these ranged from roughening of the eye to strong malformations and reduction in size .
in some cases, the eye tissue was replaced by head cuticle.
in addition, some flies exhibited a partial or total loss of ocelli .
the defects in the eye could be traced back to the imaginal discs .
the regular arrangement of the developing ommatidia was disturbed, and several additional folds and bulges were visible, both in the eye and the antennal part of the discs.
however, patterning of cells within remaining ommatidia was unaffected, as revealed by histological sections of adult eyes lacking vari function.
photoreceptor cells were correctly arranged in a trapezoid pattern as in wild-type eyes .
in order to determine the stage at which vari was required to support normal eye development, vari-rnai was induced at different developmental stages making use of a temperature-sensitive inhibitor of gal <dig>  gal80ts  <cit> .
independent of the time point of induction of vari-rnai expression , similar phenotypes with comparable penetrance and expressivity were obtained, although they were consistenly weaker than in flies that were permanently kept at 25°c .
some aspects of both the wing and the eye phenotypes obtained upon reduction of vari function by rnai are reminiscent to phenotypes observed in animals in which notch  signalling is reduced.
n is haplo-insuffient, and removal of one copy of n or expression of a dominant-negative form of its ligand serrate lead to notches in the wing  <cit> .
inactivation of the n pathway during early eye development results in reduction of eye size or complete lack of eyes  <cit> .
to test whether vari affects the activity of the notch pathway, we analysed the expression of a n activity reporter  <cit>  in wing and eye discs expressing vari-rnai.
no major changes in the overall pattern of n activity were detected, neither in the wing  nor in the eye imaginal discs , although the expression domains were often deranged/mislocalised due to the formation of extra folds and bulges, which were already visible in second instar larvae .
the only abnormality was observed at the prospective margin of wing imaginal discs, where the domain of n activation as well as expression of one of its target genes, wingless, were discontinuous in about a quarter of discs analysed .
discussion
here we demonstrate, that two out of three predicted vari proteins are in fact expressed in the embryo.
both proteins are also present in imaginal discs of third instar larvae and heads of adult flies , while in ovaries only vari-short could be detected.
in the embryo, the two isoforms are differentially expressed, the smaller one being expressed earlier and much more abundant than the larger form.
localised varicose protein is detected even later, after stage  <dig>  this, together with the analysis of vari transcripts and proteins in the different mutants suggests that there are several levels of gene expression regulation.
for example, in vari03953b the longer transcript is highly upregulated, which is not reflected at the protein level.
here, the near absence of the small isoform seems to have an effect on the synthesis and/or stability of the longer isoform.
in vari38efa <dig> mutant embryos the truncated vari-long transcript is very abundant, which is not associated with more  vari-long protein, pointing to less efficient translation an/or reduced stability of the mutant protein.
in contrast, the truncated vari-short transcript is strongly reduced in abundance, which is not paralleled by a reduction in protein levels.
in embryos mutant for this allele, properly localised protein can be detected, though in lower amounts .
since the vari antibody used does not allow discriminating between the two proteins, we cannot determine whether both isoforms are correctly localised.
although in varimd <dig>  vari <dig> or varir <dig> one or both isoforms are synthesised in normal size and amount, in no case these proteins are correctly localised at the membrane .
although not predicted by commonly used domain prediction programs, sequence comparison of vari-long with its closest vertebrate orthologues, mpp2_b, mpp6_c, and other related maguks, makes the presence of a second, more divergent n-terminal l <dig> domain in the longer vari isoform very likely.
this situation is similar to the maguk stardust, the l27n domain of which also fits less to the canonical sequence  <cit> .
the expression of two isoforms of a maguk protein, which differ by the presence or absence of the l <dig> domain is not unique.
for example, one close vari orthologue, human mpp <dig>  also encodes two isoforms, one of which, mpp6_a, lacks the two l <dig> domains .
the human postsynaptic density - <dig> protein and drosophila discs large also come in two variants, one with and one without a l <dig> domain, respectively  <cit> .
in the case of discs large, the dlga isoform is predominantly expressed in the embryo and dlg-s <dig> in the adult brain, but both isoforms are co-expressed in the larval nmj  <cit> .
in the case of vari, both isoforms are expressed in embryonic epithelia.
since the antibody used here recognises an epitope common to both vari isoforms, we cannot completely rule out the possibility that different embryonic epithelia express different vari isoforms, although this seems unlikely due to the interdependence between vari-long and vari-short.
our data further suggest that they are localised at the septate junction, since both co-immunoprecipitate with nrxiv-gfp, which is localised in the septate junction.
targeting and/or stabilisation of neurexin iv are probably mediated by direct interaction between the pdz domain of vari and the c-terminal amino acids of neurexin iv.
however, other partners of vari, particularly those interacting with the l <dig> domains, are still elusive.
similar as human vam-1/mpp6_c, which binds human veli- <dig> in vitro  <cit> , the l <dig> domain of drosophila vari can interact with the l <dig> domain of dlin- <dig> in vitro.
the different localisation of the two proteins, at least in epithelia of the drosophila embryo, however, makes their in vivo interaction in these cells very unlikely.
what could be the significance of expressing two vari isoforms?
l <dig> domains can mediate the targeting of the respective protein to a particular membrane compartment, such as the synapse or the adherens junction, or stabilise interacting proteins by directly binding to the l <dig> domain of the respective partner  <cit> .
on the other hand, maguk proteins without l <dig> domains can efficiently be brought to their proper site, using other targeting mechanisms, for example palmitoylation  <cit> .
this raises the question as to i) whether the two vari proteins rely on different mechanisms for targeting to the septate junctions and ii) whether the two vari isoforms have specific functions in the drosophila embryo.
using gal4-mediated overexpression, either of them is capable to rescue varif <dig> mutant embryos to viability.
this allele has been classified as a null allele, based on its genetic behaviour  <cit>  and the complete and nearly complete lack of vari-long and vari-short, respectively .
the rescuing capability of vari-short indicates that the l <dig> domains are not essential for viability, but does not exclude any non-essential function, in the embryo or at later stages.
strikingly, the hypomorphic allele vari38efa <dig>  which still expresses the short vari isoform, but a modified vari-long protein, gives rise to weak, but viable and fertile adults.
the deletion in this allele removes  <dig> amino acids in the n-terminus, which affects both l <dig> domains.
the fact that the escapers do not exhibit any mutant eye or wing phenotype similar to that obtained upon rnai-mediated knockdown of vari in imaginal discs suggests a more supportive function for the larger isoform.
the predominant role of vari-short is further highlighted by the fact that varimd <dig>  in which vari-short is absent, is lethal, despite expression of normal vari-long proteins.
hence, physiological amounts of only vari-long are not sufficient for viability, but excess levels and/or earlier expression  can restore viability in the absence of vari-short.
mutational analysis of vari <dig> uncovered a five amino acid deletion in the sh3-domain of vari, which almost completely removes one of the four core β-strands present in all sh- <dig> domains, thus completely abolishing vari function.
although both vari isoforms are expressed in vari <dig> in wild-type amounts, this allele is a functional null and no localised protein could be detected.
the fact that the mutant proteins are not localised suggests that either the sh <dig> domain is necessary for targeting, or that the overall structure of the protein is affected, preventing proper localisation.
structural and functional analysis of other maguks, e. g. psd- <dig> or hcask, point to either intra- or intermolecular interactions between the sh <dig> and the guk domain  <cit> .
in the maguk psd- <dig>  binding of a ligand to the pdz domain releases intramolecular inhibition of the guk domain by the sh <dig> domain  <cit> .
a strong reduction of varicose function by rnai in postembryonic stages also affects the normal development of eyes and wings.
it is interesting, however, to note that the consequences of rnai-mediated knockdown of vari in wing and eye imaginal discs seem to be different.
reduced vari function in wing imaginal discs attenuates n signaling, as revealed by lowered activity of a n reporter gene construct and disrupted expression of the n target gene wingless at the prospective margin of wing imaginal discs.
in wild-type wing imaginal discs n is activated on both sides of the dorsal/ventral compartment boundary by its two ligands, dorsally expressed serrate and ventrally expressed delta.
n activity in the wing margin activates downstream genes, like cut or wingless, which are involved in the regulation of growth and patterning .
reduction of n activity results in the formation of notches in the margin, as observed here.
in contrast, the n reporter seems to be normally activated in eye imaginal discs upon vari rnai induction.
eye imaginal discs with reduced vari function display abnormal folding and adult eyes are smaller and misshapen.
additionally, ocelli and bristles are sometimes replaced by bare head cuticle.
this phenotype is reminiscent of eye phenotypes observed in certain allelic combinations of coracle, which exhibit roughening of the eyes due to abnormally spaced ommatidia, but without affecting the patterning of the photoreceptor cells, and often lack ocelli and bristles.
in addition, some coracle mutations suppress the effects of hypermorphic mutations in the egf-receptor  <cit> .
this suggests that sjs are differentially required for normal signalling at various developmental stages, but may affect different signalling pathways in a tissue dependent way.
given that sjs are required throughout imaginal disc development as suggested by our results, their lack may affect various signalling pathways, which are spatially and developmentally regulated and integrated as shown for the egf-receptor and notch pathway  <cit> .
so far, nothing is known how sjs may affect signalling.
they could be involved in the correct localisation of receptors, membrane-bound ligands and/or components involved in signal transduction.
the stage and tissue-dependent differential contribution of various signalling pathways may explain the different phenotypes obtained upon rnai-mediated vari knockdown in eye and wing discs.
septate junctions in larval eye imaginal discs have been well documented before  <cit> , but their exact role during postembryonic development is largely unknown.
nrxiv has recently been shown to be required for septate junction formation between and among the cone and pigment cells and for ommatidial morphology and integrity  <cit> .
some of the phenotypes observed in nrxiv mutant clones in the developing eye, which are reminiscent to those obtained by eygal4-mediated overexpression of vari-rnai, have been interpreted as the result of compromised adhesion.
based on the molecular and genetic interaction between nrxiv and vari, it is tempting to speculate that vari has a comparable role during eye development.
in addition, during morphogenetic movements, cell divisions and cell rearrangements, sjs have to be redistributed  <cit> .
loss of vari may thus interfere with these processes, which could explain the abnormal folding of eygal4>uas vari-rnai eye imaginal discs.
our data suggest that the final vari mutant phenotype is the consequence of compromised sj function at different stages of development, which, in turn, may affect several cell-cell signalling and adhesion processes.
a detailed dissection of the complexity of the mutant phenotype is beyond the scope of this manuscript.
in the future it provides, however, a well-suited system to study the postembryonic function of sjs.
CONCLUSIONS
implications from our findings are threefold.
i) varicose is required for septate junction development in drosophila embryos.
ii) expression of two varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of varicose-mediated protein scaffolds at septate junctions is dynamic.
iii) varicose is required for normal wing and eye development.

 membrane-associated guanylate kinases form a family of scaffolding proteins, engaged in the organisation of multiprotein complexes, which are often associated with cellular junctions or signalling complexes, such as the vertebrate tight junction or the drosophila septate junction in epithelial cells or the neuromuscular junction. their capacity to serve as a platform for recruiting larger protein assemblies results from the presence of several protein-protein interaction domains: one to three pdz -1)-domains, an sh3--domain and a guanylate kinase -domain. some members additionally contain one or two l -domains in their n-terminus. this modular structure is ideally suited to recruit a variety of components into a protein complex, the composition of which often depends on the cell type and/or developmental stage. in addition, maguk-encoding genes often give rise to more than one isoform by alternative splicing, thus increasing the possibility of multiple interactions, localisations and/or functions. for example, the vertebrate membrane-associated palmitoylated protein encodes a retina-specific isoform, which lacks the l domain in its n-terminus. similarly, drosophila discs large expresses an isoform lacking the l domain, which is unable to bind to dlin- in epithelia, whereas an isoform containing the l domain can associate with dlin- in the neuromuscular junction. drosophila polychaetoid, the orthologue of mammalian zo- encodes two isoforms, one of which is localised apically, whereas the other one distributes more broadly along the lateral membrane of epithelial cells of the wing imaginal disc. finally, drosophila stardust expresses a distinct isoform in the embryo, characterised by the presence of a particular exon, which is not expressed in the eye. taken together, maguk proteins provide a versatile platform for the dynamic assembly of protein complexes in various cell types. septate junctions in epithelia of drosophila reside in the lateral membrane, basal to the zonula adherens. two kinds of sjs are known, pleated sjs and smooth sjs. pleated sjs are present in most ectodermally derived epithelia of the embryo, i. e. the epidermis, the tracheae, the salivary glands, the fore- and the hindgut, but they are excluded from the amnioserosa and the malpighian tubules. they are characterised by ladder-like septa spanning the space between the lateral membranes of adjacent cells. they are first visible in these epithelia around stage and become further elaborated by the end of embryogenesis. sjs can be found in epithelia of arthropods and at the blood-brain barrier of both arthropods and chordates. they serve as barriers between different compartments by providing a paracellular diffusion barrier, a function equivalent to that of vertebrate tight junctions. in addition, sjs are involved in the control of tracheal tube size. the molecular components in drosophila septate junctions include transmembrane proteins, such as neurexin iv, neuroglian, gliotactin and a na+/k+ atpase, the cell surface protein lachesin as well as scaffolding proteins, such as the ferm family member coracle and the maguk discs large. some of them are orthologues to vertebrate tj proteins, such as megatrachea or sinuous, two claudin-like proteins, or contactin. recently it has been shown that septate junctions contain an additional member of the maguk protein family, encoded by varicose, which is required for proper localisation of other septate junction components and tracheal tube size development. here we show by electron microscopy that varicose mutant embryos fail to develop proper pleated sjs. we further demonstrate that two different transcripts and proteins are expressed in the embryo, which are differentially affected by different varicose alleles, pointing to individual functions. in addition, we provide data to show that varicose is expressed in the wing and eye imaginal discs and necessary for their normal development.  varicose expresses two maguk isoforms in the embryo in drosophila epithelia, the maguk-proteins stardust and discs large are required to organise protein complexes in the subapical and the lateral membrane, respectively. sdt recruits the transmembrane protein crumbs and two other scaffolding proteins, dlin- and dpatj, into a complex, which is localised subapically, i. e. in a small region of the apical plasma membrane just apical to the zonula adherens, in epithelial and photoreceptor cells. we were interested in identifying additional partners of the crumbs complex, which might have a function in the organisation of epithelia of the drosophila embryo. therefore, we took advantage of the recently published protein interaction map to screen for partners of dlin- a component of the crumbs complex. one of the putative partners identified in this screen is encoded by drosophila cg located at 38e on the left arm of the second chromosome. it is predicted to encode three isoforms, which differ in their size and are the result of differential transcriptional initiation and alternative splicing. as demonstrated recently, these proteins are encoded by varicose and define a new subgroup of maguk proteins, which includes the mammalian proteins mpp and mpp6/vam-1/pals. previously, only a single l domain was predicted in the longer isoform. when using smart and reciprocal blasts, two l domains are predicted. this is in agreement with the domain structure of orthologues from the honeybee, mouse, xenopus and zebrafish. strikingly, human mpp also encodes two isoforms of different size, mpp6_a and mpp6_c, the longer of which carries an extended n-terminal region with two l domains. the expression of two different transcripts in the embryo was verified by northern blots and rt-pcr. using a probe common to all predicted transcripts, two mrnas of about and kb were detected on northern blots with poly-rna of staged wild-type embryos, with the shorter one being much more abundant than the longer one. the expression of the two different transcripts was confirmed by rt-pcr, using transcript-specific primers. however, rt-pcr experiments failed to detect vari transcripts encoding the predicted isoform d, suggesting that only isoforms b and c are expressed in the developing embryo, which represent the short and the long vari isoform, respectively. in-situ hybridisations with a probe common to both transcripts reveal that vari is expressed in most ectodermally derived embryonic epithelia, i. e. the epidermis, the fore- and the hindgut, the tracheae and the salivary glands, but not in the malpighian tubules. western blot analysis with antibodies raised against a recombinant protein containing the pdz- and the sh3-domain revealed that two variants of ~ and ~ kda are present in extracts of wild-type or vari-hemizygous embryos. the smaller isoform, which matches the predicted size, was already detectable in extracts from 0– hour embryos, but was much more abundant during later stages of embryogenesis. the larger isoform was only detectable in embryos older than hours, and present in only very small amounts compared to the shorter form. western blots from embryos expressing a flag-tagged vari-long protein confirmed the specificity of the larger isoform. both proteins were almost completely or completely absent in extracts of embryos expressing vari-rnai or embryos homozygous for a deficiency, which removes the entire gene, respectively. similar as shown recently, our antibody recognised an epitope in most ectodermally derived epithelia of wild-type embryos, i. e. in the epidermis, the tracheae, the salivary glands, the fore- and the hindgut, but not in the malpighian tubules. in these tissues, the protein detected by the antibody localised basal to the za, as revealed by double-staining with anti-crumbs and anti-armadillo, respectively. vari co-localised with the septate junction components neurexin iv and discs large, confirming its association with septate junctions. varicose is required for septate junction development we induced a vari mutation by imprecise excision of a p-element of the line pd, which localised immediately 5' to the transcription start site of the smaller transcript. one out of homozygous lethal excision lines isolated failed to complement deficiency dfds and vari alleles vari03953b, vari varir varir and varif. the mutant excision line carries a deletion that removes basepairs of genomic dna, thus completely eliminating exon 3a, the first exon of the transcript encoding the shorter isoform plus part of the following intron. consistent with this result, embryos homozygous mutant for this small deletion did not express any vari-short protein. the genetic and molecular data classified the mutation identified here as a vari allele, which is called varimd from here on. embryos homozygous mutant for varimd or transheterozygous for varimd109/vari03953b died at the end of embryogenesis with convoluted tracheal tubes, a phenotype similar to that described for vari03953b. apical-basal polarity was not affected, as revealed by proper apical localisation of crb. the same phenotype was achieved by specifically knocking down vari function in the tracheae by expressing vari-rnai by means of btlgal. septate junction components, such as coracle or neurexin iv, which are restricted to the lateral membrane in wild-type embryos, became distributed to all membranes in vari mutant embryos, both in the tracheae and the epidermis. this phenotype was reminiscent to that achieved in the absence of neurexin iv function. based on the failure to properly localise the septate-junction-associated proteins neurexin iv and coracle in vari mutant embryos wu et al. suggested that these embryos fail to properly establish and/or maintain the septate junctions. in order to prove this, we carried out electron microscopic analysis of wild-type and vari mutant embryos. in wild-type embryos, pleated septate junctions are clearly distinguishable from stage onwards by the presence of septa, which span the space between the lateral membranes of neighbouring cells, both in the epidermis and the tracheae. homozygous mutant varimd embryos of the same stage lacked septate junctions in these tissues. similarly, septate junctions were not detected in vari03953b homozygous mutant embryos. the za was not affected in epithelia of the mutant embryos and still formed a continuous apical belt. this result finally proves that vari has an essential function in the formation of the pleated septate junctions during embryonic development. varicose alleles differentially affect the two protein isoforms in the embryo the molecular lesions in the dna of several vari alleles, which exhibit a weak, an intermediate or a strong mutant phenotype, respectively, have been described. in none of these mutant embryos, nor in embryos mutant for varimd any localised vari protein could be detected in the embryo. we were interested to find out how the different alleles affect the expression of the two vari isoforms and whether there is any relationship between the strength of the mutant phenotype of a given allele as determined by genetic analyses and the expression of the two vari isoforms. therefore, we performed rt-pcr and western blots on extracts of homozygous mutant embryos. as expected, embryos mutant for the intermediate allele varimd in which the transcription start site of the smaller form is deleted, did not express vari-short rna or vari-short protein, but still exhibited vari-long protein expression, which seemed even slightly up-regulated. in the intermediate allele vari03953b, which carries a bp deletion in the third common intron, very low amounts of vari-short rna and protein were expressed. although vari-long transcripts were upregulated in the mutant embryos, the amount of vari-long protein was reduced in comparison to wild-type. the null allele varir carries a point mutation leading to a premature stop codon in the region encoding the hook domain, which should result in a truncation of both protein isoforms. as expected, this mutation did not affect the size of the two transcripts. western blots revealed that the long isoform was in fact truncated: a protein of about kd, corresponding to a truncated vari-long could be detected. the absence of any truncated short isoform suggested that this truncated form was not stable. the null allele varif which carries a transposon in the intron between exons 3a and did not express vari-long and only very low amounts of vari-short protein. in contrast, the insertion line pd which carries a p-element in the intron between exon and 3a expressed normal amounts of both proteins. the mutation in vari also characterised as a null allele, alters the splice acceptor site of the common exon therefore, one would expect that exon is skipped. as a consequence, both transcripts should be reduced by nucleotides and both proteins should lack amino acids. surprisingly, vari mutant embryos still expressed wild-type sized transcripts and proteins. however, sequencing the rt-pcr product obtained by amplification with primers vari-short- and vari-short- revealed, that the mutant splice site is skipped, and the next ag-site inside exon is used as a splice acceptor instead. this results in a bp deletion, which exactly removes amino acids of the sh3-domain, without affecting the reading frame. this deletion is too small to be detected in the length of the rt-pcr product or the protein. in the strong allele varir which carries a single point mutation, resulting in the exchange of an arginine to a cysteine in the hook domain, the mutation did not have any effect on the size of the two proteins, but vari-long seemed to be present in lower amount. in the weak allele vari38efa which carries a mutation in the splice acceptor site of exon the rnas of both isoforms were reduced in length, but, as expected, only the long protein isoform was truncated. taken together, the results obtained by western blot analysis showed that in most vari mutant alleles at least one of the protein isoforms is affected. varicose is necessary, but not sufficient for neurexin iv localisation the c-terminus of neurexin iv was able to pull-down the recombinant, his-tagged pdz-domain of varicose when fused to gst. we could further identify the c-terminal pdz-binding motif-eifi of neurexin iv as being crucial for this interaction, since its removal completely abolished the interaction. to confirm that these two proteins also form a complex in vivo, we made use of a protein trap line expressing a neurexin iv-gfp fusion protein, in which the p-element is inserted in the first intron and gfp is fused in frame right after the signal sequence. immunoprecipitation of neurexin iv-gfp with an anti-gfp antibody from embryonic extracts co-precipitated both varicose isoforms. it has recently been shown that neurexin iv is necessary to recruit varicose to the septate junctions. to demonstrate that it is also sufficient, we overexpressed neurexin iv in the posterior compartment of each segment, making use of an ep-insertion in the neurexin iv locus. this induced a moderate enrichment of vari protein in regions of the cell with higher nrxiv protein levels. overexpression of another sj component, dlga, failed to recruit vari to ectopic sites, further demonstrating the specificity of the nrxiv-vari interaction. in contrast, vari-long did not recruit more nrxiv to the membrane, suggesting that vari is necessary, but not sufficient for nrxiv localisation. varicose has initially been described as an interaction partner of dlin- in a yeast two-hybrid screen. this interaction could be confirmed by pull-down experiments. a gst-fusion protein, containing the l27-domain of dlin- pulled down the recombinant l domains of vari-long, fused to a his-tag. the pdz-domain of dlin- was unable to mediate this interaction. however, unlike nrxiv, overexpression of dlin- in the embryonic epidermis had no effect on the distribution of vari. varicose is required for normal wing and eye development the data show that varicose has an essential function during development of the embryo. vari protein is also expressed at later stages, in the wing and the eye imaginal discs. in the wing imaginal disc, vari is expressed throughout the disc epithelium, where it localises basal to crumbs, which marks the apical pole. in the eye disc of third instar larvae, there is enriched expression of vari behind the morphogenetic furrow. nrxiv and coracle, two other components of the septate junction, exhibit the same expression pattern, suggesting that vari is associated with sj in differentiating ommatidia. in both the eye and the wing discs, both isoforms could be detected, while ovaries expressed only vari-short, but not vari-long. to assess vari function in imaginal discs, we expressed double-stranded rna in a tissue-specific manner. overexpression of vari-rnai by means of ptcgal i. e. in a stripe of cells along the anterior-posterior compartment boundary of the wing imaginal disc, almost completely removed vari protein from these cells, suggesting that vari function was strongly suppressed. reduction of vari had no effect on apical-basal polarity as revealed by the continuous apical localisation of crumbs. use of sdgal to express vari-rnai in the developing wing pouch from first/second larval instar onwards gave rise to adult flies with defective wings or pharate adults, depending on the strength of the different vari-rnai effector-strains used. hatched flies exhibited malformations of their wings to various degrees. most prominent were gaps in the wing margin and defects in the unfolding of the wing. eygal4>uas vari-rnai animals, which expressed the vari dsrna under the control of the eyeless promotor, i. e. in the eye imaginal discs from early stages onward as well as in a small stripe in the antennal disc, developed defects in the adult eye. these ranged from roughening of the eye to strong malformations and reduction in size. in some cases, the eye tissue was replaced by head cuticle. in addition, some flies exhibited a partial or total loss of ocelli. the defects in the eye could be traced back to the imaginal discs. the regular arrangement of the developing ommatidia was disturbed, and several additional folds and bulges were visible, both in the eye and the antennal part of the discs. however, patterning of cells within remaining ommatidia was unaffected, as revealed by histological sections of adult eyes lacking vari function. photoreceptor cells were correctly arranged in a trapezoid pattern as in wild-type eyes. in order to determine the stage at which vari was required to support normal eye development, vari-rnai was induced at different developmental stages making use of a temperature-sensitive inhibitor of gal gal80ts. independent of the time point of induction of vari-rnai expression, similar phenotypes with comparable penetrance and expressivity were obtained, although they were consistenly weaker than in flies that were permanently kept at 25°c. some aspects of both the wing and the eye phenotypes obtained upon reduction of vari function by rnai are reminiscent to phenotypes observed in animals in which notch signalling is reduced. n is haplo-insuffient, and removal of one copy of n or expression of a dominant-negative form of its ligand serrate lead to notches in the wing. inactivation of the n pathway during early eye development results in reduction of eye size or complete lack of eyes. to test whether vari affects the activity of the notch pathway, we analysed the expression of a n activity reporter in wing and eye discs expressing vari-rnai. no major changes in the overall pattern of n activity were detected, neither in the wing nor in the eye imaginal discs, although the expression domains were often deranged/mislocalised due to the formation of extra folds and bulges, which were already visible in second instar larvae. the only abnormality was observed at the prospective margin of wing imaginal discs, where the domain of n activation as well as expression of one of its target genes, wingless, were discontinuous in about a quarter of discs analysed. discussion here we demonstrate, that two out of three predicted vari proteins are in fact expressed in the embryo. both proteins are also present in imaginal discs of third instar larvae and heads of adult flies, while in ovaries only vari-short could be detected. in the embryo, the two isoforms are differentially expressed, the smaller one being expressed earlier and much more abundant than the larger form. localised varicose protein is detected even later, after stage this, together with the analysis of vari transcripts and proteins in the different mutants suggests that there are several levels of gene expression regulation. for example, in vari03953b the longer transcript is highly upregulated, which is not reflected at the protein level. here, the near absence of the small isoform seems to have an effect on the synthesis and/or stability of the longer isoform. in vari38efa mutant embryos the truncated vari-long transcript is very abundant, which is not associated with more vari-long protein, pointing to less efficient translation an/or reduced stability of the mutant protein. in contrast, the truncated vari-short transcript is strongly reduced in abundance, which is not paralleled by a reduction in protein levels. in embryos mutant for this allele, properly localised protein can be detected, though in lower amounts. since the vari antibody used does not allow discriminating between the two proteins, we cannot determine whether both isoforms are correctly localised. although in varimd vari or varir one or both isoforms are synthesised in normal size and amount, in no case these proteins are correctly localised at the membrane. although not predicted by commonly used domain prediction programs, sequence comparison of vari-long with its closest vertebrate orthologues, mpp2_b, mpp6_c, and other related maguks, makes the presence of a second, more divergent n-terminal l domain in the longer vari isoform very likely. this situation is similar to the maguk stardust, the l27n domain of which also fits less to the canonical sequence. the expression of two isoforms of a maguk protein, which differ by the presence or absence of the l domain is not unique. for example, one close vari orthologue, human mpp also encodes two isoforms, one of which, mpp6_a, lacks the two l domains. the human postsynaptic density - protein and drosophila discs large also come in two variants, one with and one without a l domain, respectively. in the case of discs large, the dlga isoform is predominantly expressed in the embryo and dlg-s in the adult brain, but both isoforms are co-expressed in the larval nmj. in the case of vari, both isoforms are expressed in embryonic epithelia. since the antibody used here recognises an epitope common to both vari isoforms, we cannot completely rule out the possibility that different embryonic epithelia express different vari isoforms, although this seems unlikely due to the interdependence between vari-long and vari-short. our data further suggest that they are localised at the septate junction, since both co-immunoprecipitate with nrxiv-gfp, which is localised in the septate junction. targeting and/or stabilisation of neurexin iv are probably mediated by direct interaction between the pdz domain of vari and the c-terminal amino acids of neurexin iv. however, other partners of vari, particularly those interacting with the l domains, are still elusive. similar as human vam-1/mpp6_c, which binds human veli- in vitro, the l domain of drosophila vari can interact with the l domain of dlin- in vitro. the different localisation of the two proteins, at least in epithelia of the drosophila embryo, however, makes their in vivo interaction in these cells very unlikely. what could be the significance of expressing two vari isoforms? l domains can mediate the targeting of the respective protein to a particular membrane compartment, such as the synapse or the adherens junction, or stabilise interacting proteins by directly binding to the l domain of the respective partner. on the other hand, maguk proteins without l domains can efficiently be brought to their proper site, using other targeting mechanisms, for example palmitoylation. this raises the question as to i) whether the two vari proteins rely on different mechanisms for targeting to the septate junctions and ii) whether the two vari isoforms have specific functions in the drosophila embryo. using gal4-mediated overexpression, either of them is capable to rescue varif mutant embryos to viability. this allele has been classified as a null allele, based on its genetic behaviour and the complete and nearly complete lack of vari-long and vari-short, respectively. the rescuing capability of vari-short indicates that the l domains are not essential for viability, but does not exclude any non-essential function, in the embryo or at later stages. strikingly, the hypomorphic allele vari38efa which still expresses the short vari isoform, but a modified vari-long protein, gives rise to weak, but viable and fertile adults. the deletion in this allele removes amino acids in the n-terminus, which affects both l domains. the fact that the escapers do not exhibit any mutant eye or wing phenotype similar to that obtained upon rnai-mediated knockdown of vari in imaginal discs suggests a more supportive function for the larger isoform. the predominant role of vari-short is further highlighted by the fact that varimd in which vari-short is absent, is lethal, despite expression of normal vari-long proteins. hence, physiological amounts of only vari-long are not sufficient for viability, but excess levels and/or earlier expression can restore viability in the absence of vari-short. mutational analysis of vari uncovered a five amino acid deletion in the sh3-domain of vari, which almost completely removes one of the four core β-strands present in all sh- domains, thus completely abolishing vari function. although both vari isoforms are expressed in vari in wild-type amounts, this allele is a functional null and no localised protein could be detected. the fact that the mutant proteins are not localised suggests that either the sh domain is necessary for targeting, or that the overall structure of the protein is affected, preventing proper localisation. structural and functional analysis of other maguks, e. g. psd- or hcask, point to either intra- or intermolecular interactions between the sh and the guk domain. in the maguk psd- binding of a ligand to the pdz domain releases intramolecular inhibition of the guk domain by the sh domain. a strong reduction of varicose function by rnai in postembryonic stages also affects the normal development of eyes and wings. it is interesting, however, to note that the consequences of rnai-mediated knockdown of vari in wing and eye imaginal discs seem to be different. reduced vari function in wing imaginal discs attenuates n signaling, as revealed by lowered activity of a n reporter gene construct and disrupted expression of the n target gene wingless at the prospective margin of wing imaginal discs. in wild-type wing imaginal discs n is activated on both sides of the dorsal/ventral compartment boundary by its two ligands, dorsally expressed serrate and ventrally expressed delta. n activity in the wing margin activates downstream genes, like cut or wingless, which are involved in the regulation of growth and patterning. reduction of n activity results in the formation of notches in the margin, as observed here. in contrast, the n reporter seems to be normally activated in eye imaginal discs upon vari rnai induction. eye imaginal discs with reduced vari function display abnormal folding and adult eyes are smaller and misshapen. additionally, ocelli and bristles are sometimes replaced by bare head cuticle. this phenotype is reminiscent of eye phenotypes observed in certain allelic combinations of coracle, which exhibit roughening of the eyes due to abnormally spaced ommatidia, but without affecting the patterning of the photoreceptor cells, and often lack ocelli and bristles. in addition, some coracle mutations suppress the effects of hypermorphic mutations in the egf-receptor. this suggests that sjs are differentially required for normal signalling at various developmental stages, but may affect different signalling pathways in a tissue dependent way. given that sjs are required throughout imaginal disc development as suggested by our results, their lack may affect various signalling pathways, which are spatially and developmentally regulated and integrated as shown for the egf-receptor and notch pathway. so far, nothing is known how sjs may affect signalling. they could be involved in the correct localisation of receptors, membrane-bound ligands and/or components involved in signal transduction. the stage and tissue-dependent differential contribution of various signalling pathways may explain the different phenotypes obtained upon rnai-mediated vari knockdown in eye and wing discs. septate junctions in larval eye imaginal discs have been well documented before, but their exact role during postembryonic development is largely unknown. nrxiv has recently been shown to be required for septate junction formation between and among the cone and pigment cells and for ommatidial morphology and integrity. some of the phenotypes observed in nrxiv mutant clones in the developing eye, which are reminiscent to those obtained by eygal4-mediated overexpression of vari-rnai, have been interpreted as the result of compromised adhesion. based on the molecular and genetic interaction between nrxiv and vari, it is tempting to speculate that vari has a comparable role during eye development. in addition, during morphogenetic movements, cell divisions and cell rearrangements, sjs have to be redistributed. loss of vari may thus interfere with these processes, which could explain the abnormal folding of eygal4>uas vari-rnai eye imaginal discs. our data suggest that the final vari mutant phenotype is the consequence of compromised sj function at different stages of development, which, in turn, may affect several cell-cell signalling and adhesion processes. a detailed dissection of the complexity of the mutant phenotype is beyond the scope of this manuscript. in the future it provides, however, a well-suited system to study the postembryonic function of sjs.  implications from our findings are threefold. i) varicose is required for septate junction development in drosophila embryos. ii) expression of two varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of varicose-mediated protein scaffolds at septate junctions is dynamic. iii) varicose is required for normal wing and eye development. 
the two isoforms can be distinguished by the presence or absence of two l domains and are differentially affected in different varicose alleles. here we show by electron microscopic analysis that drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. postembryonic reduction of varicose function by expressing double-stranded rna affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes. in the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the maguk family. their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. the two proteins interact in vitro by the pdz domain of varicose and the four c-terminal amino acids of neurexin iv. varicose proteins co-localise with neurexin iv in pleated septate junctions and are necessary, but not sufficient for its recruitment. membrane-associated guanylate kinases form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the drosophila septate junction or the neuromuscular junction. expression of two varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function. while the short isoform is essential for viability, the long isoform seems to have a supportive function.
 membrane-associated guanylate kinases form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the drosophila septate junction or the neuromuscular junction. their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. they often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes.  here we show by electron microscopic analysis that drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. in the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the maguk family. the two isoforms can be distinguished by the presence or absence of two l domains and are differentially affected in different varicose alleles. while the short isoform is essential for viability, the long isoform seems to have a supportive function. varicose proteins co-localise with neurexin iv in pleated septate junctions and are necessary, but not sufficient for its recruitment. the two proteins interact in vitro by the pdz domain of varicose and the four c-terminal amino acids of neurexin iv. postembryonic reduction of varicose function by expressing double-stranded rna affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.  expression of two varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function. 