InteractiveFly: GeneBrief

serpentine and vermiform: Biological Overview | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

The function of tubular epithelial organs like the kidney and lung is critically dependent on the length and diameter of their constituting branches. Genetic analysis of tube size control during Drosophila tracheal development has revealed that epithelial septate junction (SJ) components and the dynamic chitinous luminal matrix coordinate tube growth. However, to date the underlying molecular mechanisms controlling tube expansion have remained elusive. Two luminal chitin binding proteins, Serpentine and Vermiform, possessing predicted polysaccharide deacetylase activities (ChLDs) are required to assemble the cable-like extracellular matrix (ECM) and restrict tracheal tube elongation. Overexpression of native, but not of mutated, ChLD versions also interferes with the structural integrity of the intraluminal ECM and causes aberrant tube elongation. Whereas ChLD mutants have normal SJ structure and function, the luminal deposition of the ChLD requires intact cellular SJs. This identifies a new molecular function for SJs in the apical secretion of ChLD and positions ChLD downstream of the SJs in tube length control. serp and verm are not required for chitin synthesis or secretion but rather for its normal fibrillar structure. They are responsible for the N-deacetylation of chitin and conversion of chitin to chitosan, a polysaccharide composed of repeating glucosamine units; obtained by de-acetylation of chitin. The mutations also affect structural properties of another chitinous matrix, epidermal cuticle. In tracheal development, deposition of the chitin luminal matrix first promotes and coordinates radial tube expansion. It is proposed that the subsequent structural modification of chitin by chitin binding deacetylases selectively instructs the termination of tube elongation to the underlying epithelium (Wang, 2006; Luschnig, 2006).

An important late step in the morphogenesis of tubular organs is the final acquisition of distinct branch sizes. The fixed length and diameter of tubes dictate the flow rates of the transported gas or liquid and are therefore major determinants of optimal organ function. Several of the genes controlling tube size during organogenesis encode potential regulators of apical membrane and cytoskeletal organization, but the cellular mechanisms measuring and restricting lumen growth are not well understood. In the Drosophila airways, a dynamic chitinous extracellular matrix forms inside the developing branches and is required for uniform tube expansion. Loss of the transient chitin cable results in cystic and winding tubes and defects in apical cell shapes and cytoskeletal organization, suggesting that the expanding polysaccharide cable controls tube extension. How does the luminal chitin matrix signal its structural integrity to the epithelium? The lamellar structure and stiffness of arthropod cuticles depends on the arrangement of chitin polymers [β(1-4)-linked N-acetylglucosamine, GlcNAc] in planes of parallel or antiparrallel fibrils through the formation of hydrogen bonds between the chains (Merzendorfer, 2003). Moreover, the rigidity of the Saccharomyces cerevisiae spore cell wall requires chitin deacetylases that convert chitin to chitosan, a β(1-4) N-Glucosamine (GlcN) polymer with distinct physical properties. It is hypothesized that similar luminal chitin modifications resulting in structural changes of the matrix may control tube length or diameter growth. The fly genome encodes more than 100 putative chitin binding proteins (ChtB, IPR002557; Magkrioti, 2004), and three of them, encoded by CG8756, CG32209, and CG17905, contain additional Low-Density Lipoprotein Receptor (LDLR, IPR002172) and polysaccharide deacetylase (Polysacc Deac, IPR002509) domains, suggesting that they may be involved in the assembly and maturation of chitin polymers. This protein family has been named ChLDs because of the predicted structure of the encoded proteins, and the CG8756 and CG32209 members were named vermiform (verm) and serpentine (serp), respectively, because of the tracheal phenotypes of the mutants. The expression patterns of verm, serp, and ChLD3 were examined in wild-type embryos by in situ hybridizations. verm and serp are expressed in all tracheal cells from stage 12 until the end of embryogenesis, and both genes are also expressed in the epidermis from stage 15 onward. ChLD3 expression was not detected in the trachea, and it was therefore not analyzed further. To investigate the localization of the Verm protein, antibodies were raised against its C terminus and wild-type embryos were stained. Verm was detected inside all tracheal cells from stage 12. Verm deposition into the lumen of the dorsal trunk (DT) commences abruptly at stage 14, followed by all other branches thereafter. By stage 15, the Verm labeling was luminal and lining the apical cell surface of the DT. This staining was severely reduced to background levels in verm mutants. btl-GAL4 was used to express Serp-GFP fusion protein and it was found to be secreted into the lumen similarly to endogenous verm (Wang, 2006).

The dynamic localization of ChLD proteins as well as their predicted structure suggests a role in the assembly of the intraluminal chitinous filament. To determine the function of Verm and Serp, strong loss-of-function mutations were obtained for both genes. Stage-16 verm mutant embryos stained for the luminal 2A12 antigen display excessive elongation and convolutions of the DTs, which become gradually more pronounced in later stages. This defect becomes largely ameliorated in mutants expressing UAS-verm in the trachea, suggesting that verm restricts tube elongation. Similarly elongated DT tubes were detected in serp mutant embryos. In addition, Df(3L)Exel6135 embryos, lacking ten genes including verm and serp, show more severe tube-overextension phenotypes than either single mutant. These mutants also displayed a slight diametric overgrowth in the DT, suggesting that the removal of both ChLDs, or other genes in their neighborhood, may also influence the restriction of radial tube growth. The analysis of verm and serp embryos labeled with anti-DE-Cadherin antibodies also revealed overstretched cellular profiles. This indicates that both verm and serp are required to halt tube elongation and co-ordinate apical cell extensions at late stage 15. The aggravated phenotype of embryos deficient for both proteins suggests that they may cooperate in that task (Wang, 2006).

Do the tube-length defects in the mutants correlate with phenotypes in the assembly and structure of the luminal matrix? Luminal chitin assembly was analyzed by a fluorescent chitin binding protein (ChtB) and a fluorescent chitin binding lectin (WGA) (Tonning, 2005). Both reagents revealed a cylindrical structure of tightly packed luminal chitin polymers in the wild-type. In homozygous verm and hemizygous serp embryos, this structure appeared diffuse and expanded radially already at stage 15. By stage 16, its labeling intensity was severely reduced in the DTs compared to the wild-type. Analysis of transverse transmission electron microscopy (TEM) sections of wild-type DTs at stage 16 reveals a homogenously packed luminal matrix of parallel fibrils. The orientation and assembly of the fibrils is distorted in verm mutants, indicating that ChLDs organize intraluminal chitin packing. The formation of the luminal matrix depends on the chitin synthetase encoded by krotzkopf verkehrt (kkv) (Ostrowski, 2002). In kkv mutants, the chitinous cable is absent and the tubes first fail to expand their diameter and later become over elongated and convoluted (Tonning, 2005). The tube phenotypes of kkv verm double mutants were identical to the ones of kkv, indicating that chitin synthesis is a prerequisite for ChLD function. In addition, the luminal Verm staining was severely disrupted in kkv mutants. Whereas early events of tracheal cuticle assembly, like the apical deposition of the epicuticle layers at early stage 16, were unaffected in verm mutants, cuticular abnormalities were detected in DT cross-sections at late stage 16. These include irregular taenidia, reduced procuticle deposition, and aberrant apical membranes, revealing an additional, later role of Verm in tracheal cuticle assembly. The above data argue that ChLDs are primarily required to form or preserve the tight texture of the chitinous luminal cable and that their function and localization require kkv and its product chitin (Wang, 2006).

To further analyze the functional potential of ChLD proteins, native and GFP-tagged versions of Verm and Serp were overexpressed in the trachea and the phenotypes were assessed in embryos stained for the ChtB reagent or 2A12. Expression of either transgene caused distortion of the filamentous texture and a reduction in the staining intensity of the chitin cable. Whereas native Verm causes lesser effects, Serp and both GFP-tagged versions induce more-pronounced phenotypes. Moreover, the tube-overgrowth and -winding phenotypes in the tracheal branches were proportional to the severity of the intraluminal matrix defect. Thus, ChLD-induced changes in filament structure play an instructive role in tube elongation (Wang, 2006).

To dissect the function of the three predicted domains of Serp in this assay, transgenic embryos were generated expressing GFP-tagged versions lacking each of the motifs. Tracheal expression of Serp-ΔChtB-GFP or Serp-ΔLDLR-GFP did not cause any detectable luminal or tube-extension phenotype, indicating that both domains are necessary for the function of Serp in tube length control. Expression of Serp-ΔDeac-GFP did not affect tube elongation either, but, surprisingly, it was found that the protein does not become secreted but accumulates inside the tracheal cells. Thus, the deacetylase domain harbors motifs required for Serp luminal secretion. The failure of the deletion constructs to cause phenotypes argues against antimorphic effects of the ChLD-GFP proteins and indicates that all three predicted domains are required for tracheal Serp function. Overexpression of the ChLDs leads to similar luminal and tube-elongation phenotypes as their loss of function, suggesting that both lack of as well as increased levels of chitin modifications result in similar effects (Wang, 2006).

The analysis of several Drosophila mutants with overgrown tracheal tubes similar to verm and serp embryos demonstrated that paracellular septate junction (SJ) components are required for halting lumen overgrowth. Two different molecular mechanisms have been proposed to account for the tube-overextension phenotypes in SJ mutants: (1) SJ-associated proteins like Scribble and Discs Large may restrict the activity of apical-membrane growth-promoting factors like Crb and DaPKC during tube growth, and (2), SJ functions may be required in luminal matrix assembly and thereby indirectly regulate tube expansion (Tonning, 2005). Is there a mechanistic link between the function of SJs and ChLDs in tube length control? Whether ChLDs are required for the assembly of SJs was examined. Df(3L)Exel6135 and verm mutant embryos were initially examined in three different assays. (1) Staining was performed using antibodies against the SJ markers Neurexin and FasIII; (2) DT crossections of verm mutants were analyzed for the ladder-like SJ structure by TEM; (3) the function of the paracellular barrier was tested by Dextran injections. ChLD mutants were indistinguishable from wild-type in all three experiments, arguing that ChLD does not affect SJ structure or function. Is Verm affected in SJ mutants? The localization of Verm was analyzed in mutants for the α subunit of the Na/K-ATPase (Atpα). In these embryos, Verm is retained inside the tracheal cells at stage 15, whereas its luminal abundance is strongly reduced and becomes gradually undetectable at stage 16. This defect in Verm luminal secretion is accompanied by only a subtle decrease in the luminal 2A12 staining. The same phenotypes were detected of aberrant Verm intracellular accumulation and strongly reduced luminal levels in sinu (Claudin) and lac (bulb) mutants, suggesting that Verm luminal secretion depends on the assembly or maintenance of the SJ structure. The defect of Verm intracellular retention and luminal stabilization in SJ mutants is unlikely to reflect a general block of apical secretion because neither the 2A12 marker nor the luminal ZP-protein Pio showed defects in luminal accumulation at stage 15. The distinct luminal-deposition phenotypes demonstrate a new function for the transepithelial barrier junctions in the apical secretion of Verm. Moreover, these apical secretion defects provide molecular evidence that ChLDs act downstream of the SJs to regulate tube size. The sorting signal required for apical targeting via SJs is presently unknown, and its identification may be aided by the dissection of the role of sequences in the Deacetylase domain in luminal secretion (Wang, 2006).

The phenotypic analysis of known tracheal-tube-size mutants unveiled an instrumental role of the chitinous extracellular matrix structure in orchestrating branch dimensions. The assembly and growth of the chitin cable first coordinates the radial expansion of the tubes. Subsequent modifications in chitin fibril structure by secreted ChLDs instruct the underlying epithelium to terminate tube elongation. Thus, dynamic structural changes of the luminal matrix may be sensed independently to determine diametric growth and tube elongation. Given the predicted structure of ChLDs, such changes may involve chitin deacetylation and consequently the extent of hydrogen bonding between chitin fibers and their packing. The similar loss- and gain-of-function ChLD phenotypes suggest that the distinct level of chitin modification and the expected changes in matrix rigidity monitor tube elongation. Luminal fibrillar ECM structures have been described in a vertebrate angiogenesis model, and this genetic analysis provides a first mechanistic view on distinct functions of the luminal ECM in tube size control (Wang, 2006).

The two genes define a new molecular class of tube morphogenesis genes encoding apical extracellular matrix proteins that modify the chitin matrix. Both proteins contain chitin binding and deacetylase domains, and both are secreted into the apical tracheal matrix, where they associate with, and modify the structure of, the chitin cylinder that fills most of the luminal space. In serp verm double mutants, the chitin cylinder still forms, but it lacks its normal fibrillar appearance. The chitin cylinder, along with the surrounding lumen and apical tracheal surface, becomes excessively long and convoluted, a process that normally occurs gradually over the next hours and days of development as the tubes expand to their mature sizes and acquire their characteristic shapes (Luschnig, 2006).

The results, along with the recent identification of chitin biosynthesis genes in tracheal-tube morphogenesis, demonstrate the dual and genetically separable functions of chitin in tracheal tube diameter and length control. The chitin synthesis genes cystic, knk/gnarled, and kkv chitin synthase are required to synthesize the expanding chitin cylinder in the tracheal lumen, which is proposed to promote and coordinate the dilation of the surrounding epithelium so that tubes reach their proper diameter. By contrast, the chitin modification genes serp and verm are not required for synthesis of chitin and have little effect on dilation and tube diameter. Rather, they influence the structure of the chitin cylinder and the length and curvature of the tubes. The chitin cylinder may therefore function as an internal template that plays a critical role in defining the diameter, length, and shape of the tube that surrounds it. A molecular model is presented for the role of Serp and Verm proteins and the chitin cylinder in tube lengthening and it is suggested how this mechanism could be regulated to control the longitudinal growth of tracheal tubes during development (Luschnig, 2006).

There are four postulates of the model. (1) It is proposed that the Serp and Verm proteins bind and modify chitin; (2) this modification alters the physical properties of the chitin cylinder, keeping it rigid and short; (3) these changes in the chitin cylinder are sensed by the surrounding tracheal cells, perhaps through a direct link between an apical-membrane component and a constituent of the chitin cylinder; (4) this signal restricts apical-membrane biogenesis, in a manner that limits polarized growth of the cell membrane specifically along the longitudinal axis of the tube (Luschnig, 2006).

Much of the data supports the first two postulates. Serp and Verm proteins are secreted into the tracheal lumen, where they associate with the chitin cylinder, an association that is likely mediated by the chitin binding domain. The chitin modification is most probably deacetylation of N-acetylglucosamine residues by the chitin deacetylase domain of the proteins. This is a well-known structural modification of chitin in yeast and fungi, and the enzymatic catalysis of this reaction has received much attention because of the commercial use of deacetylated chitin (chitosan) in water treatment, in the food industry, and in medical applications such as fabrication of artificial skin. Deacetylation increases the solubility and decreases the density of chitin fibrils in vitro, and it may influence the structure and orientation of chitin fibrils in arthropod cuticle. This apparently increases the rigidity of the chitin matrix, as implied by the defect in cell wall rigidity in a yeast mutant lacking chitin deacetylase activity and by the lax epidermal cuticle in serp verm double mutants (Luschnig, 2006).

The last two postulates of the model are more speculative. It is not known if or how the chitin cylinder is attached to the apical cell surface. There is a characteristic ~0.5 μm gap that is between the cell surface and the chitin cylinder and is visible in fixed specimens and could contain an anchoring complex. The gap is eliminated in serp verm double mutants. How the proposed link between chitin and the apical cell surface, or a signal generated by this complex, limits apical-membrane biogenesis along the longitudinal axis of the tube is even more obscure. Perhaps it exerts a mechanical effect on the apical cell surface, physically constraining apical membrane elongation, or maybe it influences the distribution or activity of apical-basal cell-polarity regulators such as Crumbs. Whatever the mechanism, the genetic results make clear that absence of Serp and Verm proteins results in a dramatic expansion of the lumen and apical tracheal surface -- but only along the longitudinal axis of the tube (Luschnig, 2006).

Longitudinal growth of tracheal tubes normally occurs gradually and continuously during development, beginning soon after the tubes form during embryogenesis and continuing throughout larval life. This allows the size and transport capability of the tracheal network to keep pace with the increasing oxygen demand of the growing larva. Because this growth occurs in the absence of tracheal cell division and is only periodically interrupted by a burst of radial growth, it must involve the gradual and continuous expansion of tracheal cells specifically in the longitudinal axis of the tube. This is reminiscent of the effects of serp and verm mutations in the embryo, except that in the mutants, tube elongation occurs more precipitately than normal. Because the serp and verm genes are expressed early and broadly in the developing tracheal system, under control of Ribbon and possibly Trachealess, they could act as governors on tube growth from the onset, keeping tube elongation in check. Controlled downregulation of Serp and Verm expression or activity during development could gradually release this constraint and give rise to the controlled longitudinal tube growth that is normally observed. Septate-junction mutants have a tube-elongation phenotype similar to that of serp and verm mutants, so septate junctions could function to promote Serp and Verm expression or activity or to antagonize the negative regulatory pathway (Luschnig, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

mRNAs of serpentine and vermiform are detected in the developing tracheal system beginning at embryonic stage 12. Both genes are also expressed in the developing stomodeum beginning at stage 14 and in the epidermis at stage 16. Tracheal expression of both genes starts to fade at stage 15 and is no longer detected at stage 16 (Luschnig, 2006).

Antisera were generated against short synthetic peptides derived from the serp and verm coding sequences, respectively. Immunostaining and immunoblot analysis demonstrated that the anti-Serp and anti-Verm antisera do not cross-react with one another's proteins. Serp and Verm proteins are first detected in early stage-13 embryos in a punctate perinuclear distribution in tracheal cells. About 1 hr later, just before Dorsal trunk (DT) branches fuse, the two proteins begin to accumulate in the lumen of the DT. They appear first in the lumen of the most posterior DT segments and slightly later in more anterior DT segments. During stages 14 and 15, the proteins appear in the lumen of all of the other tracheal branches. Intracellular staining of Serp and Verm proteins persists through stage 15, but is no longer detected at stage 16 (Luschnig, 2006).

Chitin forms a cylinder inside the tracheal lumen, and this cylinder is first detected in the DT just before DT dilation and then expands as the lumen dilates. Luminal chitin is secreted hours before the chitinous tracheal procuticle forms at the end of embryogenesis, and it is degraded or expelled from the tracheal lumen when the tubes are mature and just before they fill with gas. The chitin cylinder has a slightly smaller diameter than that of the lumen, which is bounded by the apical surface of the tracheal epithelium. Strong staining of Serp and Verm protein colocalized with the chitin cylinder, whereas there was only weak staining in the small gap between the cylinder and the apical surface of DT cells. This contrasts with the distribution of another luminal antigen, 2A12, which was distributed throughout the entire luminal space including the gap (Luschnig, 2006).

The colocalization of Serp and Verm with the chitin cylinder, and the presence in each protein of a chitin binding domain (CBD), suggest that the proteins directly associate with the chitin cylinder. Two experiments support this. (1) The organized and regular distribution of Serp and Verm proteins in the lumen requires chitin. In embryos homozygous for kkv¹ (a mutation in the chitin synthase I gene), which lack tracheal chitin, Serp and Verm proteins are still expressed and secreted into the lumen. However, the distribution of the proteins is altered -- they form irregular, amorphous masses in the lumen, as does 2A12 antigen. (2) The CBD is apparently sufficient for localization to the chitin cylinder. A transgene expressing just an N-terminal fragment of Serp including the CBD fused to GFP [Serp(CBD)-GFP showed the same colocalization with the chitin cylinder as endogenous Serp protein and full-length Serp-GFP. It is concluded that Serp and Verm proteins associate with the luminal chitinous matrix and that this association is likely to be mediated at least in part by the CBD (Luschnig, 2006).

Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis

Epithelial tubes are the functional units of many organs, but little is known about how tube sizes are established. Using the Drosophila tracheal system as a model, it has been shown that mutations in varicose (vari) cause tubes to become elongated without increasing cell number. This study shows how vari is required for accumulation of the tracheal size-control proteins Vermiform and Serpentine in the tracheal lumen. vari is an essential septate junction (SJ) gene encoding a membrane associated guanylate kinase (MAGUK). In vivo analyses of domains important for MAGUK scaffolding functions demonstrate that while the Vari HOOK domain is essential, the L27 domain is dispensable. Phylogenetic analyses reveal that Vari helps define a new MAGUK subgroup that includes mammalian PALS2. Importantly, both Vari and PALS2 are basolateral, and the interaction of Vari with the cell-adhesion protein Neurexin IV parallels the interaction of PALS2 and another cell-adhesion protein, Necl-2. Vari therefore bolsters the similarity between Drosophila and vertebrate epithelial basolateral regions, which had previously been limited to the common basolateral localization of Scrib, Dlg and Lgl, proteins required for epithelial polarization at the beginning of embryogenesis. However, by contrast to Scrib, Dlg and Lgl, Vari is not required for cell polarity but rather is part of a cell-adhesion complex. Thus, Vari fundamentally extends the similarity of Drosophila and vertebrate basolateral regions from sharing only polarity complexes to sharing both polarity and cell-adhesion complexes (Wu, 2007).

The function of organs such as the lung, kidney and vascular system depends on epithelial and endothelial tubes of specific sizes. However, the cell biological and molecular processes that control tube sizes are largely unknown. The Drosophila tracheal system is a network of ramifying epithelial tubes that serves as a combined pulmonary-vascular system to directly deliver oxygen to tissues. The comparative simplicity and genetic tractability of the tracheal system has made it one of the best models of tubular epithelial morphogenesis. The tracheal system develops from a series of sacs into a complex network of branches through a highly orchestrated series of cell migrations, cell shape changes and rearrangements of cell-cell junctions. An important element of these morphogenetic events is that changes in tube size occur reproducibly during specific developmental periods. Each tracheal branch has a specific size that results from the action of branch-specific signaling events that at least in some branches are known to act through transcription factors such as Spalt-Major (Spalt). At least one additional transcription factor, Grainyhead, is required to control tube length and apical cell surface in the major tracheal branches, but the transcriptional targets that more directly mediate these functions remain to be identified. Recent work by multiple groups has produced a basic molecular framework of the mechanisms that execute the size changes of 'tube expansion', a process that increases the diameter - but not the length - of the major tracheal tubes over a 2 hour period, and then gradually lengthens the tubes without changing their diameters. These tube size changes result from changes in cell shape and possibly cell size, but do not involve changes in cell number (Wu, 2007).

The tube expansion mechanism depends upon a fibrillar, chitin-based extracellular matrix that is assembled in the tracheal lumen at the beginning of the diameter dilation. As development progresses, chitin at the apical cell surface is organized into a highly patterned, multilayered cuticle. Lumenal chitin is eliminated before hatching. Defects in chitin synthesis or organization cause tracheal tube diameters to become either too large or too small, and tube lengths to become over-elongated. The exact role of the chitin-based matrix in controlling tracheal cell shape is unclear. Although the lumenal matrix and cuticle may serve as structural forms or 'mandrils' that mechanically shape the tracheal cells and tubes, an instructive or signaling role for the matrix is suggested by the observation that the organization of the β_H-spectrin cytoskeleton is altered in chitin-synthetase mutants (Wu, 2007).

Beginning at stage 15, organization of the lumenal matrix requires the lumenal secretion of the putative chitin deacetylases, Vermiform (Verm) and Serpentine (Serp). In verm and serp mutants, the chitin-based matrix becomes disorganized and tracheal tubes become too long. Surprisingly, lumenal secretion of Verm requires a cell-cell junction termed the septate junction (SJ). Septate junctions are complex cell adhesion junctions that have at least 15 known components. These include transmembrane cell-adhesion proteins such as Neurexin IV (Nrx-IV; herein referred to as Nrx) and Neuroglian (Nrg), cytoplasmic proteins such as the FERM-domain protein Coracle (Cor; Cora - Flybase), the basal polarity proteins Scribbled (Scrib), Discs large (Dlg; Dlg1 -Flybase), and Lethal giant larvae (Lgl; L(2)gl - Flybase), and proteins with roles that remain to be determined, such as the Na+/K+-ATPase. Mutations in most known SJ components cause tracheal phenotypes indistinguishable from the verm mutant phenotype, consistent with the failure of Verm to be secreted into the tracheal lumen in the SJ mutants so far examined. Secretion of other apical lumenal markers appears normal in SJ mutants, indicating that Verm is secreted by a specialized pathway, the mechanism of which remains to be determined (Wu, 2007).

Although the role of SJs in lumenal (apical) secretion is not understood, other SJ functions are well defined. SJs have functional and molecular similarity to vertebrate tight junctions (TJs), in that both junctions require members of the claudin protein family to create the paracellular diffusion barriers between epithelial cells that are essential to the survival of multicellular animals. However, SJs are not simply the homologs of TJs, because there are significant ultrastructural, molecular and functional differences between SJ and TJs. For example, TJs are apical of adherens junctions (AJs) and contain conserved apical polarity complexes, while SJs are basal of AJs and contain the polarity proteins Scrib, Dlg and Lgl, which have vertebrate homologs that also localize basolaterally. Thus, in some respects SJs are more related to complexes found in the basolateral regions of vertebrate epithelial cells than to TJs (Wu, 2007).

Although Scrib, Dlg and Lgl establish and currently define the similarity between SJ and vertebrate basolateral regions, it is notable that these proteins are not representative of most SJ components. Drosophila Scrib, Dlg and Lgl are maternally contributed and constitute a distinct subgroup of proteins required for initial epithelial cell polarization during embryonic stages 5-8. By contrast, most SJ components are not maternally expressed, are not required for cell polarity and only function relatively late in development when SJs begin forming during stage 13. Whether the Scrib, Dlg and Lgl proteins nucleate SJ assembly, or whether the nascent SJ recruits and incorporates Scrib, Dlg and Lgl has not been determined. It also has not yet been determined how Scrib, Dlg and Lgl are localized to the basolateral membrane in either Drosophila or vertebrate epithelia. Thus the similarity between Drosophila SJ and vertebrate basolateral regions has been limited to polarity complexes, and has not extended to cell adhesion complexes (Wu, 2007).

This report shows that vari encodes a previously uncharacterized, membrane-associated, guanylate kinase (MAGUK) scaffolding protein that is required for SJ organization and directly binds the cell adhesion protein Neurexin IV. Importantly, Vari helps define a new subgroup of MAGUKs that includes vertebrate PALS2. Both Vari and PALS2 localize basolaterally in epithelial cells and both interact through a PDZ domain with a basolateral adhesion protein. Thus, Vari is the first late-expressed SJ component to have a vertebrate homolog, and together Vari and PALS2 extend the similarity of Drosophila and vertebrate basolateral regions from polarity complexes to adhesion complexes (Wu, 2007).

Vari was originally identified as a gene required for regulating the size of epithelial tubes. In vari mutants, tracheal tubes become too long without changes in tracheal cell number. This study shows that Vari encodes multiple isoforms of a MAGUK that helps define a new subgroup of MAGUKs. Vari functions in the assembly of the septate junctions and is required for the apical secretion of the protein Verm, which is thought to be responsible for modifying a chitin-based lumenal matrix. In vari and other SJ mutants, Verm is not secreted, the lumenal matrix becomes abnormal and tracheal tubes become elongated (Wu, 2007).

The protein-protein interaction domains present in Vari suggest it acts as a scaffolding protein that helps bring together different components of the SJ complex. This hypothesis is supported by GST-pull down assay results showing Vari's PDZ domain can directly bind the intracellular domain of Nrx, a transmembrane SJ adhesion protein. Binding of the Vari PDZ domain to Nrx would leave Vari's SH3, GUK and predicted C-terminal PDZ-binding motif available to anchor other SJ components to the membrane, or to bring together different transmembrane SJ components. One model is that Vari may help bring the Dlg-Scib complex to the membrane through interfolding of the Vari and Dlg SH3 domains, which is made possible by the unique HOOK domain insert in the MAGUK SH3 domains. Whether or not Vari anchors the Dlg complex to the rest of the SJ, genetic evidence indicates that Vari has functions beyond simply bridging between transmembrane Nrx and intracellular SJ complexes, because vari mutations can strongly enhance the phenotypes caused by mutations in the Drosophila claudin sinuous, whereas nrx mutations do not enhance sinuous mutations (Wu, 2007).

By itself, the finding that Vari encodes a MAGUK was not unexpected, as many MAGUKs are associated with cell-cell junctions. However, it is significant that Vari helps define a new subgroup of MAGUKs that includes mammalian PALS2, because Vari and PALS2 both localize basolaterally and bind the C-termini of basolateral cell adhesion proteins. Thus, Vari and PALS2 bolster the similarity between Drosophila and vertebrate epithelial basolateral regions that was first evidenced by the common basolateral localization of the Scrib, Dlg and Lgl early polarity proteins. However, by contrast to the polarity proteins, Vari is not required for cell polarity but rather is expressed late in embryonic development and is part of a cell-adhesion complex. Thus, Vari fundamentally extends the similarity of Drosophila and vertebrate basolateral regions from containing only conserved polarity complexes to containing both conserved polarity and cell-adhesion complexes (Wu, 2007).

The finding of more extensive similarity between SJ and vertebrate basolateral regions suggests that continued study of Drosophila SJs will provide insight into vertebrate epithelial basolateral regions. Further, these results support the idea that during evolution there has been conservation of different junctional functions, such as forming paracellular barriers and anchoring of polarity complexes. However, the comparison of TJs and SJs also makes it clear that there has been limited conservation of which particular functions have assorted to different junctions. An attractive explanation for these somewhat contradictory observations is that junctional functions are modular, and that the disparate junctions in different species represent alternative combinations of functional modules. For example, Drosophila SJs could be considered a combination of the claudin-based paracellular-barrier function and the basolateral polarity proteins Dlg, Scrib and Lgl. Alternatively, vertebrate TJs could be considered a combination of the claudin-based paracellular-barrier function and the apical polarity complexes of Crbs-Baz and Sdt-aPKC-Par-6. Thus, when comparing junctions between species, it is likely to be more useful to compare specific junctional functions, such as molecular details of polarity or barrier functions, than to attempt to directly compare junctions in their entirety (Wu, 2007).

If complex junctions such as TJs and SJs are comprised of functional modules, one would expect that these junctions should contain distinct molecular subcomplexes that mediate distinct functions. Consistent with this proposal, extensive work by many labs has shown that the polarity proteins of Crb-Sdt and Baz-cdc42-aPKC form specific complexes. Claudin proteins appear to be part of a 'barrier complex' because claudins are required for and co-localize with the paracellular barrier in both Drosophila and vertebrates. Functional demonstration of the independence of the barrier and polarity complexes in both species is provided by the observations that cell polarity is not affected by selective disruption of the barrier complex in either mammals by knockdown of ZO-1 and ZO-2, or in Drosophila by mutations in claudin genes. The Vari/PALS2 proteins could play a pivotal role in allowing cytoplasmic subcomplexes to associate different adhesion-junctional complexes, either in different cell types or during evolution, because changing which adhesion complex Vari or PALS2 associate with could be as simple as changing the four amino acid PDZ-binding motifs of one or a few transmembrane proteins. It seems likely that evolving a few unstructured amino acids would be significantly easier than evolving three-dimensional binding surfaces. Thus, Vari and its homologs could provide crucial (but malleable) links between conserved intracellular complexes and the divergent transmembrane junctional complexes found across the animal kingdom (Wu, 2007).

A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila

The apical extracellular matrix plays a central role in epithelial tube morphogenesis. In the Drosophila tracheal system, Serpentine (Serp), a secreted chitin deacetylase expressed by the tracheal cells plays a key role in regulating tube length. This study shows that the fly fat body, which is functionally equivalent to the mammalian liver, also contributes to tracheal morphogenesis. Serp is expressed by the fat body, and the secreted Serp is taken up by the tracheal cells and translocated to the lumen to functionally support normal tracheal development. This process is defective in rab9 and shrub/vps32 mutants and in wild-type embryos treated with a secretory pathway inhibitor, leading to an abundant accumulation of Serp in the fat body. Fat body-derived Serp reaches the tracheal lumen after establishment of epithelial barrier function and is retained in the lumen in a chitin synthase-dependent manner. These results thus reveal that the fat body, a mesodermal organ, actively contributes to tracheal development (Dong, 2014).

EFFECTS OF MUTATION

Many organs contain epithelial tubes that transport gases or liquids. Proper tube size and shape is crucial for organ function, but the mechanisms controlling tube diameter and length are poorly understood. Recent studies of tracheal (respiratory) tube morphogenesis in Drosophila show that chitin synthesis genes produce an expanding chitin cylinder in the apical (luminal) extracellular matrix (ECM) that coordinates the dilation of the surrounding epithelium (Tonning, 2005; Devine, 2005). This study describes two genes involved in chitin modification, serpentine (serp) and vermiform (verm), mutations that cause excessively long and tortuous tracheal tubes. The genes encode similar proteins with an LDL-receptor ligand binding motif and chitin binding and deacetylation domains. Both proteins are expressed and secreted during tube expansion and localize throughout the lumen in a chitin-dependent manner. Unlike previously characterized chitin pathway genes, serp and verm are not required for chitin synthesis or secretion but rather for its normal fibrillar structure. The mutations also affect structural properties of another chitinous matrix, epidermal cuticle. This work demonstrates that chitin and the matrix proteins Serp and Verm limit tube elongation, and it suggests that tube length is controlled independently of diameter by modulating physical properties of the chitin ECM, presumably by N-deacetylation of chitin and conversion to chitosan (Luschnig, 2006).

Genetic pathways controlling branching morphogenesis and cell-type diversification of the Drosophila tracheal system have been characterized. However, it is not known how tracheal cells measure, regulate, and maintain distinct sizes and shapes of epithelial tubes. Genetic screens have identified genes that influence the diameter, length, and shape of tracheal tubes (Beitel, 2000). Many of these encode components of septate junctions, the insect cognate of vertebrate tight junctions. Recently, genes involved in the synthesis of a cylindrical chitin matrix secreted by tracheal cells prior to cuticle formation were identified and shown to play an essential role in controlling tracheal tube diameter (Devine, 2005; Tonning, 2005). This study described the identification and characterization of two genes that encode apical extracellular matrix (ECM) proteins that modify the structure of the chitin matrix and regulate tracheal tube length (Luschnig, 2006).

In a genomic search for genes regulated by the transcription factor Ribbon, a nuclear BTB/POZ domain protein that promotes movement and morphogenesis of the apical surface of the tracheal epithelium, two adjacent genes were identified at cytological position 76C1-2 (CG32209 and CG8756) that encode structurally related tracheal matrix proteins. The genes are expressed in indistinguishable patterns. mRNAs of both genes were detected in the developing tracheal system beginning at embryonic stage 12, just after Ribbon protein is detected, and expression of both genes was reduced in ribbon null mutants as determined by in situ hybridization and DNA microarray analysis (6-fold reduction of CG32209 and 5-fold reduction of CG8756). The genes were named serpentine (serp; CG32209) and vermiform (verm; CG8756) on the basis of their elongated and convoluted tracheal phenotypes described below (Luschnig, 2006).

To analyze the developmental functions of the genes, putative null mutations in each gene were identified. A P element insertion in CG8756 (verm^KG) is embryonic lethal when homozygous. Excision of the transposon restored viability of the parental chromosome in 9 of 13 excision events, indicating that the lethality is due to the P element. One imprecise excision, verm^ex7, removed the transposon and 556 bp of flanking genomic DNA, including the first coding exon of CG8756, which is common to all known splice variants and includes the start codon and signal peptide, suggesting that verm^ex7 is a null allele. The parental P element insertion also appears to be a null allele because Verm protein expression was not detected by immunostaining and its phenotype was indistinguishable from verm^ex7. serp^RB is an insertion in CG32209 of a PiggyBac transposon designed to disrupt mRNA splicing. It eliminated expression of Serp protein and behaved as a null allele in genetic tests (Luschnig, 2006).

Tracheal development in serp and verm mutants was analyzed with specific markers for tracheal cells and the tracheal lumen. No defects were detected early in tracheal development in any of the homozygous mutants analyzed. Dorsal trunk (DT) branches budded, fused, and dilated normally. However, during stage 15 (~13 hr after egg lay at 25°C) in both serp and verm homozygous embryos, the DT began to elongate inappropriately and became convoluted. The effects are more dramatic in homozygous serp^RB verm^KG double mutants: The DT began to elongate excessively at stage 15 and by stage 16 (15 hr AEL) was 40% longer than normal and highly convoluted. Similar effects were observed in other branches including the transverse connective (TC), although the effects were not as pronounced in smaller-caliber branches, such as the dorsal branch (DB). The phenotype of hemizygous serp^RB verm^KG embryos (in trans to Df(3L)Exel6135 that removes serp and verm) was indistinguishable from homozygous serp^RB verm^KG embryos. In contrast to the dramatic effects of the mutations on tracheal tube length and shape, there was little or no effect of the mutations on the diameter of the tubes. The DT showed its characteristic posterior to anterior taper, and was of normal caliber except for slight constrictions that were occasionally observed near DT fusion joints in serp^RB verm^KG double mutants. It is concluded that serp and verm are required to restrict tracheal tube length. This distinguishes them from a second class of tracheal-tube morphogenesis genes that are required to establish and maintain correct tube diameter and are involved in chitin synthesis (e.g., kkv) (Luschnig, 2006).

In serp^RB verm^KG double mutants, the tracheal lumen is excessively long and forms dramatic corkscrew-like twists. Immunostaining for Crumbs (Crb) protein, which localizes to the apical marginal zone of epithelial cells, showed that the apical tracheal surface is similarly elongated and convoluted in the mutants. However, the basal (outer) surface of the tracheal epithelium did not appear to follow the convoluted path of the lumen and apical surface. This suggests that serp and verm act to selectively restrict expansion of the lumen and apical surface of the tracheal epithelium. A similar mutant phenotype has been described for grainyhead (grh), which encodes a transcription factor proposed to restrict tracheal tube elongation through transcriptional regulation of apical matrix genes (Hemphala, 2003). Serp and Verm proteins are still expressed in grh^IM mutant embryos, suggesting that serp and verm are not critical targets of Grh (Luschnig, 2006).

The selective effect of serp and verm mutations on the apical surface and the length and convolution of tracheal tubes also resembles the tube-morphogenesis defect of mutants in megatrachea and other genes that encode components of septate junctions (SJs). Although the mechanism by which SJs influence tube length is not understood, all of the SJ mutants that have been tested affect the pericellular-diffusion barrier function of tracheae and other epithelia. To determine whether tracheal barrier function is compromised in serp verm double mutants, rhodamine-labeled dextran (MW ~10 kDa) was injected into the body cavity of mutant and control embryos and its distribution was analyzed 25 min later. In mega^G0012 and other SJ mutants, dextran passes through the tracheal epithelium and into the lumen. By contrast, in serp verm double mutants, dextran is excluded from the tracheal lumen, as it is in the wild-type control. It is concluded that epithelial barrier function is grossly intact in the serp verm double mutant and that the tube-morphogenesis defect does not result from disruption of SJ barrier function (Luschnig, 2006).

Whether serp and verm mutations affect the synthesis or structure of chitin was investigated. There was no detectable effect in serp verm double-mutant embryos on the level of luminal chitin staining, demonstrating that serp and verm function is not required for the synthesis, secretion, or luminal accumulation of chitin. Likewise, the secretion and luminal accumulation of 2A12 antigen and the zona pellucida protein PioPio were not disrupted in serp verm mutants. However, the morphology and structure of the luminal chitin cylinder was altered in serp verm mutant embryos. High-resolution confocal imaging of luminal chitin stained with a fluorescently labeled chitin binding protein revealed that the chitin cylinder in wild-type embryos is fibrous and has a smooth surface. By contrast, in serp verm mutants, the fibrous structure of the chitin cylinder is abolished and the surface of the cylinder is irregular. Also, the small gap between the chitin cylinder and the apical epithelial surface is absent. Morphological defects in chitin structure are apparent in serp verm mutants by stage 14, several hours before the elongated-tube phenotype begins to manifest. This implies that the defects in chitin structure are not a secondary consequence of the disruption in tube morphology, and support an alternative model in which a serp- and verm-dependent alteration in chitin structure influences tube length. serp and verm are also expressed in epidermal cells, and the mutations affect body shape, presumably by altering the structure and rigidity of epidermal cuticle, another chitinous matrix (Luschnig, 2006).

These findings in the Drosophila tracheal system could have implications for the mechanisms of tube size and shape regulation in other tubular epithelial organs, including those of vertebrates. For example, blood vessels grown in vitro from human endothelial cells contain a fibrous luminal matrix that is of unknown composition and function and has been postulated to play a role in tube morphogenesis. Although there are many molecular differences among the luminal ECMs of blood vessels, tracheal tubes, and other tubular organs in animals, they could act similarly to regulate and maintain the diameter, length, and shape of the surrounding tubes. Indeed, synthetic mandrils are used in this way in blood-vessel engineering. It will be important to identify and characterize the components of the luminal matrices of blood vessels and other types of tubes, and to determine whether dynamic and specific changes in the structure and physical properties of these matrices are used to regulate tube size and shape in vivo, as proposed for Drosophila tracheal tubes (Luschnig, 2006).

REFERENCES

Search PubMed for articles about Drosophila serpentine and vermiform

Beitel, G. J. and Krasnow, M. A. (2000). Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127(15): 3271-82. 10887083

Devine, W. P., Lubarsky, B., Shaw, K., Luschnig, S., Messina, L. and Krasnow, M. A. (2005). Requirement for chitin biosynthesis in epithelial tube morphogenesis. Proc. Natl. Acad. Sci. 102(47): 17014-9. 16287975

Dong, B., Miao, G. and Hayashi, S. (2014). A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila. Development 141: 4104-4109. PubMed ID: 25336738

Hemphala, J., Uv, A., Cantera, R., Bray, S. and Samakovlis, C. (2003). Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling. Development 130(2): 249-58. 12466193

>Luschnig, S., Batz, T., Armbruster, K. and Krasnow, M. A. (2006). serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila. Curr. Biol. 16(2): 186-94. 16431371

Magkrioti, C. K., et al. (2004). cuticleDB: a relational database of Arthropod cuticular proteins. BMC Bioinformatics 5: 138. 15453918

Merzendorfer, H. and Zimoch, L. (2003). Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J. Exp. Biol. 206(Pt 24): 4393-412. 14610026

Ostrowski, S., Dierick, H. A. and Bejsovec, A. (2002). Genetic control of cuticle formation during embryonic development of Drosophila melanogaster. Genetics 161(1): 171-82. 12019232

Tonning, A., Hemphala, J., Tang, E., Nannmark, U., Samakovlis, C. and Uv, A. (2005). A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea. Dev. Cell 9(3): 423-30. 16139230

Tsigos, I., Martinou, A., Kafetzopoulos, D. and Bouriotis, B. (2000). Chitin deacetylases: New, versatile tools in biotechnology, Trends Biotechnol. 18: 305-312. 10856926

Wang, S., Jayaram, S. A., Hemphala, J., Senti, K. A., Tsarouhas, V., Jin, H. and Samakovlis, C. (2006). Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea. Curr. Biol. 16(2): 180-5. 16431370

Wu, V. M., et al. (2007). Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis. Development 134(5): 999-1009. PubMed Citation: 17267446

date revised: 12 January 2018

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