InteractiveFly: GeneBrief

Mummy: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Tracheal and nervous system development are two model systems for the study of organogenesis in Drosophila. In two independent screens, three alleles were identified of a gene involved in tracheal, cuticle and CNS development. These alleles, and the previously identified cystic and mummy, all belong to the same complementation group. These are mutants of a gene encoding the UDP-N-acetylglucosamine diphosphorylase, an enzyme responsible for the production of UDP-N-acetylglucosamine, an important intermediate in chitin and glycan biosynthesis. cyst was originally singled out as a gene required for the regulation of tracheal tube diameter. The cyst/mmy tracheal phenotype was identified and upon histological examination it was concluded that mmy mutant embryos lack chitin-containing structures, such as the procuticle at the epidermis and the taenidial folds in the tracheal lumen. While most of their tracheal morphogenesis defects can be attributed to the lack of chitin, when compared to krotzkopf verkehrt (kkv) chitin-synthase mutants, mmy mutants showed a stronger phenotype, suggesting that some of the mmy phenotypes, like the axon guidance defects, are chitin-independent. These data have implications in the mechanism of size control in the Drosophila trachea (Araujo, 2005). The mmy mutant phenotype is similar to that of the so-called 'Halloween' mutants, which fail to produce the differentiation hormone 20-Hydroxyecdysone (Gilbert, 2004), and whose role during insect embryogenesis remains an enigma. Mummy functions in apical extracellular matrix formation by producing GlcNAc residues needed for chitin synthesis and protein glycosylation, and dynamic mummy expression is hormonally regulated in apical extracellular matrix differentiating tissues (Tonning, 2006).

mummy is also required for epidermal cutical formation. Compared with the wild-type larval cuticle, the cuticle of larvae harbouring a strong mmy allele is hardly visible, whereas larvae mutant for the weak mmy allele develop a bloated cuticle and a deformed and strongly melanised head skeleton. mmy mutant and wild-type larval epidermis were compared by transmission electron microscopy (TEM). Wild-type cuticle is composed of three layers: (1) the outermost envelope characterised by five alternating electron-dense and electron-lucid sheets, (2) the underlying epicuticle built up by an upper electron-lucid and a lower electron-dense sublayer, and (3) the innermost procuticle structured by lamellar chitin microfibrils and contacting the apical plasma membrane of the epidermal cells. All three cuticle layers are affected in mutant mmy larvae. The outer envelope is thinner than in the wild type with only three sheets, and the electron-dense sub-layer of the epicuticle disintegrates and spreads into the upper electron-lucid sub-layer and the procuticle. The procuticle is also reduced in thickness and seems to be devoid of chitin microfibrils; occasionally, the cuticle detaches from the epidermal surface. The cuticle of larvae mutant for the weak mmy allele is stratified as in the wild type, and the procuticular chitin microfibrils appear correctly oriented. However, the procuticle of weak mutants contains abnormal inclusions of electron-dense material that are scattered below the epicuticle, presumably orphan proteins, suggesting that the coordinated assembly of the epi- and pro-cuticle is impaired. Taken together, this evidence shows that cuticle assembly requires mmy activity (Tonning, 2006).

The Drosophila tracheal system has proven to be a particularly appropriate model for the study of tubulogenesis. The larval tracheal system of Drosophila is a complex tubular network that conducts oxygen from the exterior to the internal tissues. It arises from the tracheal placodes, clusters of ectodermal cells that appear at each side of 10 embryonic segments, from the 2nd thoracic segment to the 8th abdominal segment. The cells of each cluster invaginate and migrate in a stereotypic pattern to form each of the primary tracheal branches. The general conclusion from many studies is that the direction of migration of the tracheal cells relies on a set of positional cues provided by nearby cells. In addition, the establishment of interactions between tracheal cells and their substrates is a crucial step in tracheal cell migration, a process ultimately determined by molecules expressed at their surface (Araujo, 2005).

Genetic analyses have identified many genes required for specific steps of tracheal morphogenesis, such as tube fusion and cell intercalation during formation of finer branches. One of the features of the tracheal system is that the tubes in each branch have specific sizes and diameters that appear to be precisely regulated during development. Several genes have been reported to affect the size of the tracheal tubes. Among these, a group of genes originally identified as controlling tube length have been found to code for proteins belonging to or associated with the septate junctions (SJs). Another gene, cystic (cyst), was previously singled out as being specifically required for the regulation of tracheal tube diameter (Beitel, 2000). This study reports the identification of further alleles of cystic; that cyst is allelic to the previously identified mummy (mmy) gene (Nüsslein-Volhard, 1984); that cyst/mmy is required for cuticle formation and the morphogenesis of the central nervous system (CNS), and that it encodes the only predicted Drosophila melanogaster UDP-N-acetylglucosamine diphosphorylase (UDP-GlcNAc diphosphorylase; also named UDP-N-acetylglucosamine pyrophosphorylase). This enzyme is required for the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), a substrate for chitin and glycan synthesis (Herscovics, 1993; Merzendorfer, 2003). Accordingly, it is shown that cyst/mmy is required for chitin deposition in the trachea and for the formation of the embryonic cuticle (Araujo, 2005; Tonning, 2006). Finally, the tracheal defects associated with the cyst/mmy mutant phenotype are described and the implications on the mechanism of tracheal tube size control are discussed (Araujo, 2005).

UDP-GlcNAc diphosphorylase catalyzes the formation of UDP-GlcNAc, which is essential for chitin synthesis, membrane biosynthesis, protein N- and O-glycosylation and GPI anchor biosynthesis. This enzyme is well conserved and has clear homologues across different species. The human orthologue of the Drosophila gene is UAP1, which has been shown to be expressed in human sperm and to be the antigen responsible for antibody-mediated human infertility (Diekman, 1994). In S. cerevisiae, ScUAP1 deletions are lethal and mutants display an aberrant morphology (Mio, 1998; Mio, 1999). In the genome of D. melanogaster, Mummy is the only predicted UDP-GlcNAc diphosphorylase. Another enzyme involved in the UDP-GlcNAc metabolism is the UDP-GlcNAc epimerase that interconverts UDP-GlcNAc and UDP-GalNAc. This enzyme could provide an alternative route to UDP-GlcNAc synthesis and explain the relative mildness of the phenotypes in the absence of such a fundamental enzyme as UDP-GlcNAc diphosphorylase. However, there is no predicted UDP-GlcNAc epimerase in D. melanogaster. In view of the importance of UDP-GlcNAc diphosphorylase for the synthesis of UDP-GlcNAc and the ubiquitous requirement for this metabolite, the relatively mild phenotypes and the survival of these embryos until later stages is attributed to the presence of a strong maternal contribution (Araujo, 2005).

The embryonic phenotypes for the mmy mutations arise as a consequence of the dwindling amounts of available UDP-GlcNAc. The production of different UDP-GlcNAc requiring molecules in different tissues is likely to exhibit variable sensitivity to the loss of UDP-GlcNAc diphosphorylase activity. The phenotypes observed may be due to the combined reduction of several UDP-GlcNAc containing products or primarily due to a lack of one particular molecule. The tracheal and cuticle phenotypes are principally due to the lack of chitin. This absence of chitin is not responsible for the CNS phenotype present in mmy embryos; this defect is not present in mutants for the chitin synthase CS-1 (Ostrowski, 2002; Moussian, 2005a). The CNS phenotype is likely to be due to a deficit in the appropriate glycosylation of one or more molecules. Normal development of the nervous system requires cellular interactions such as recognition and adhesion as well as the ability to send and receive signals. Many of these signaling interactions are mediated by glycoproteins, glycolipids and proteoglycans and GPI-linked proteins all of which would be affected by the reduction or absence of UDP-GlcNAc. The fidelity of axon fasciculation is known to be affected by alterations to glycan expression, and carbohydrate binding proteins are required for accurate CNS development. GlcNAc is also a major constituent of the glycosaminoglycans that are added to heparan sulfate proteoglycans (HSPGs), which are required for multiple signaling pathways. The activity of Slit, a key midline derived signaling molecule that directs axon extension both across the midline and fascicle choice by longitudinal axons in Drosophila, is modulated by the HSPGs, Syndecan and Dallylike and that axon sorting in Zebrafish requires HSPG synthesis. Additionally, it has been suggested that an appropriate pattern of HSPGs is necessary for axons to select their appropriate pathways. This study finds that loss of UDP-GlcNAc diphosphorylase activity affects axon pathway choice. Future work utilizing genetic interactions should identify which products become depleted to give rise to this CNS phenotype (Araujo, 2005).

The tracheal system of mmy mutant embryos appears to develop normally until the stage of tube formation. Even at later stages when these embryos are severely disrupted, the overall organization of the tracheal cells appears normal, at least in terms of their apical basal polarity and the restricted expression of the other proteins analyzed. Yet, at the same later stages, the general arrangement of the tracheal lumen is severely distorted. Noticeably, in mature mmy embryos, the luminal envelope is detached from the tracheal cell membrane. This emphasizes the fact that the proper tubular structure and its interaction with the surrounding cells can play an important role in maintaining the general constitution of the tracheal system following tube formation (Araujo, 2005).

Secretion of luminal components is an important step during tube formation and expansion. Vesicle-like structures have been reported to be involved both in tube expansion and in cuticle formation at the epidermis. During cuticle formation, microvillae are detected at the epidermal cell membranes prior to the formation of the cuticular envelope, and chitin is believed to be delivered to the cell surface via vesicles that fuse with the plasma membrane. In mmy mutants, as in kkv, only the chitin-free envelope and the epicuticle is detected, because the chitin-rich procuticle is never synthesized. Failure to deliver chitin to the cell surface and the subsequent lack of the procuticle both in the trachea and in the epidermis result in the detachment of cells from the chitin-free cuticular structures, thereby affecting luminal and cuticle stability (Araujo, 2005).

This contribution of the lack of chitin to the mmy phenotype is confirmed by the comparative analysis with kkv mutants. However, the kkv phenotypes constitute only a subset of those displayed by mmy. Detailed examination of kkv mutants indicates chitin-independent defects in the mmy tracheal system, particularly in what relates to the lack of lumen continuity of the dorsal trunk. In addition, the zygotic expression of mmy begins earlier (at stage 11) than kkv (at stage 13) (Moussian, 2005a), long before chitin is synthesized in the tracheal lumen (Araujo, 2005).

An additional lack of GlcNAc containing proteins at the cell surface or within the extracellular matrix could further affect the luminal stability in mmy embryos. In wild-type, at the site of fusion, after the fusion cells from adjacent metameres have made contact and the cadherin rings form, a lumen is formed inside at the junction between these cells. This lumen further expands to give rise to a continuous tube, and the tripartite cadherin remains at the site of fusion. In mmy embryos, the fusion cells seem to be properly determined and to express adequate fusion markers, but a continuous lumen is rarely achieved. The observed defects could be due to structural problems aggravated by the absence of GlcNAc either in the tracheal lumen or in the structure of the cadherin ring itself. Additionally, as in the CNS, mmy tracheal defects not present in kkv could partly arise as a consequence of the impairment of a signaling process mediated by GlcNAc containing proteins. GlcNAc is a major component of glucosaminoglycan chains attached to heparan sulfate proteoglycans (HSPGs). HSPGs play a major role in multiple signaling pathways involving Wingless, Hedgehog, FGF or Decapentaplegic (Araujo, 2005).

A remarkable feature of the dorsal trunk of mmy embryos is the absence of taenidial folds, the annular rings around the tracheal lumen. Since these structures are thought to provide some stiffness to the tracheal tubes, their absence could have an important influence in the irregular diameter of the dorsal trunk. Considering that during these developmental stages the tracheal lumen is filled with liquid, regions of prominent expansions could reflect the lack of rigidity of the tubes. In combination with the failure to establish proper lumen continuity at the fusion points, lack of rigidity could be an important factor contributing to the overall bubble-like structure of mmy dorsal trunks. Finally, accumulation of Pio luminal protein seems to be unaffected in mmy mutants, as opposed to the accumulation of the lumen epitope recognized by the 2A12 antibody, suggesting that not all the luminal components are impaired in mmy mutants and that different luminal structures appear to be specified independently (Araujo, 2005).

Different branches of the tracheal system have specific and distinct diameters and lengths. These features are very stereotyped and have been suggested to be under the control of a genetic program. Indeed, many genes have been unveiled that, when mutated, produce enlargements or expansions of the tubes. Some of these genes have been recently characterized and, despite being originally identified as controlling tube length, have been found to code for proteins belonging to or associated with the septate junctions (SJs). However, besides their effect on tube length, mutations in these genes also cause a failure in the trans-epithelial diffusion barrier. Among the genes influencing tube size, cyst/mmy has been singled out as a diameter-specific regulatory gene (Beitel, 2000; Wu, 2004). Shown here is evidence that the tracheal tube expansions, constrictions and consequent diameter variations in mmy mutants reflect a severe disorganization of lumen structure. In fact, many of the tracheal branches of the mmy mutants have lost their tubular characteristic and form collapsed, independent, vesicle-like structures. Thus, besides affecting tube diameter, the mmy gene is involved in the proper organization of the tracheal cells and tracheal luminal cuticle, and the expansions and constrictions seem to be side effects of disrupting these events (Araujo, 2005).

The above-mentioned observations suggest that many of the genes that have been ascribed to the genetic control of tube size may simply be required for cell arrangement, proper tube fusion and/or physiological and cuticle organization of the tracheal tube epithelia. In this regard, mmy, kkv and even the SJ mutants do not appear to modify only the tube size itself, but also its organization, bringing into doubt whether there is a specific genetic size-control program. Conversely, it is suggested that many features of tube size might not be under the independent control of a specific genetic program but, instead, that size may be a structural property of the organization of each specific branch. According to this view, the size control of a particular tube would not be something imposed upon a branch but rather a consequence of its cellular organization. For example, while in some branches the surfaces of two or more cells contribute to the luminal circumference ('multicellular tubules'), in most branches, the tube circumference is made from single cells wrapped around the lumen ('unicellular tubules'). Consistently, 'multicellular tubules' are wider than 'unicellular tubules', and it has been recently shown that the latter are originated by cell intercalation, a process that is under genetic control. Thus, tube diameter could be indirectly controlled by the program regulating cell intercalation. Similarly, tracheal cell shapes are very different in the branches formed along the anteroposterior axis, compared to the ones formed along the dorsoventral axis; the former ones adopt an elongated shape, while the latter remain cuboidal. Since these cell shapes are also related to the basic organization of the different tracheal branches, they could also contribute to the final length of the tubes. Again, this difference in cell shape is regulated by the specific signaling pathways responsible for the migration in one or the other axis. Thus, once the basic organization of the distinct branches is set, the remaining process of lumen formation and the final thickness of the tracheal epithelium could be a determinant for the final size of the tubes (Araujo, 2005).

Finally, the basic features of the specific branches are also determined by the constraints of the surrounding tissues. (1) The dynamic expression of the Branchless (Bnl) chemoattractant molecule determines the final position acquired by the tracheal branches and (2) the topological constraints will also have a role in the process. Thus, for instance, development of the dorsal trunk requires the existence of a population of lateral mesoderm cells that act as a substrate for migration of the tracheal cells forming this branch, whereas formation of the dorsal branch requires tracheal cell migration through a groove of muscle precursor cells of defined width. In summary, it is suggested that many features of tube size are not under the independent control of a specific genetic program but instead are derived from both the surrounding constraints and the distinct organization properties of each particular branch (Araujo, 2005).

REGULATION

Tramtrack regulates different morphogenetic events during Drosophila tracheal development

Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, a detailed analysis of Ttk function was undertaken. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, it was found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, Ttk was defined as the first identified regulator of intracellular lumen formation and; it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development (Araújo, 2007).

As with the transcription factors Trh and Vvl, which are involved in orchestrating early events of tracheal development, Ttk plays a role in orchestrating several late tracheal events. Ttk69 has been found to act mostly as a repressor. This study identified Ttk targets that appear to be negatively regulated (such as mummy (mmy), encodes a UDP-N-acetylglucosamine pyrophosphorylase enzyme required for the synthesis of the building blocks of chitin, and escargot (esg) whereas others appear to be positively regulated (such as polychaetoid (pyd) and branchless (bnl). In this latter case, Ttk might be converted into a positive regulator, as already described during photoreceptor development (Araújo, 2007).

This study identified multiple tracheal requirements for Ttk. Interestingly, most of them depend on Ttk regulating events downstream of cell fate specification, at the level of cellular responses. Additionally, a few other requirements depend on cell fate specification, as has been described for most other functions of Ttk in other developmental situations. For instance, Ttk regulates fusion cell specification by acting as a target and mediator of Notch, as occurs during sensory organ development and oogenesis. Such regulation of Ttk by N might be post-transcriptional, as occurs during sensory organ development. Remarkably, it was found that, although Ttk is sufficient to repress esg expression in fusion cells, it might not be the only esg- and fusion fate-repressor, because absence of Ttk does not increase the number of Esg-positive cells, as does downregulating N. Other N targets might be redundant with Ttk, and such redundancy could reinforce N-mediated repression of fusion fate in positions in which inductive signals (such as Bnl, Dpp and Wg) are very high, particularly near the branch tips (Araújo, 2007).

Cell rearrangements during development are common to most animals and ensure proper morphogenesis. During tracheal development, many branches grow and extend by cell intercalation. Several cellular and genetic aspects of tracheal intercalation have been well described. However, targets of Sal (which inhibits intercalation) are currently unknown (Araújo, 2007).

This study identified Ttk as a new and positive regulator of intercalation. Ttk is involved in cell junction modulation by transcriptionally regulating pyd, the only junctional protein shown, so far, to affect intercalation. In fact, modulation of AJs has been proposed to play a role during intercalation. However, Pyd cannot be the only Ttk effector of intercalation, because the pyd mutant phenotype is much weaker than that of ttk mutants. Accordingly, it was found that, in ttk mutants, cells in branches that usually intercalate remain paired and cuboidal, and appear unable to change shape and elongate. Although other explanations could account for the impaired intercalation detected in ttk mutants, it is proposed that inefficient cell shape changes represent the main cause, and might prevent the proper accomplishment of several events, such as the sliding of cells, formation of a first autocellular contact and zipping up, thereby blocking intercalation. Hence, it is proposed that cell shape changes, particularly cell elongation, are an obligate requisite for different steps of intercalation. Other targets of Ttk might presumably be regulators or components of the cytoskeleton involved in cell shape changes. It is relevant to point out here that Ttk has also been proposed to regulate morphogenetic changes required for dorsal appendage elongation (Araújo, 2007).

How does Ttk relate to the known genetic circuit (Sal-dependent) involved in intercalation? Being a transcription factor, Ttk initially appeared as an excellent candidate to participate in this genetic network by regulating sal and/or kni expression. However, both these genes to be normally expressed in ttk mutants, and several differences were detected in the intercalation phenotype of ttk loss versus sal upregulation. For instance, although both situations block intercalation, cells expressing sal, unlike those lacking ttk, are still able to undergo a certain change in shape, from cuboidal to elongated. Therefore, the results fit a model in which Ttk acts in a different and parallel pathway to Sal during intercalation. Consistent with this model, it was found that Ttk is not sufficient to promote intercalation on its own, because its overexpression cannot overcome the inhibition of intercalation imposed by Sal in the DT. Finally, genetic interactions also favour this model, because it was found that: (1) ttk overexpression did not rescue lack of intercalation produced by sal overexpression (even though it rescued the intercalation defects of ttk mutants), and (2) absence of sal (by means of the constitutive activation of the Dpp pathway) does not overcome the intercalation defects of ttk mutants. Therefore, it is proposed that Ttk promotes intercalation by endorsing changes in cell shape, but absence of Sal is still required to allow other aspects of intercalation to occur (Araújo, 2007).

Tube size regulation is essential for functionality. It was found that Ttk is involved in such regulation. Tube expansion and extension relies on a luminal chitin filament that assembles transiently in the tracheal tubes. The metabolic pathway that leads to chitin synthesis involves several enzymes, among which are Mmy and krotzkopf verkehrt (Kkv, a Chitin synthase). In addition, other proteins are known to participate in the proper assembly and/or modification of the chitin filament, such as Knk, Rtv, Verm and Serp. SJs are also required to regulate tube size and it was proposed that they exert this activity, at least partly, via the control of the apical secretion of chitin modifiers. The current results revealed that ttk acts as a key gene in tube size control, playing at least two roles: it regulates chitin filament synthesis and septate junction (SJ) activity (Araújo, 2007).

SJ regulation by Ttk appears functional rather than structural: mild defects were detected in the accumulation of only some SJ markers and there was a loss of the transepithelial diffusion barrier, whereas accumulation of other markers and SJ localisation remained apparently unaffected. It is speculated that Ttk transcriptionally controls one or several SJ components that contribute to maintain the paracellular barrier and to control a specialised apical secretory pathway. As a result, chitin binding proteins such as Verm or Serp are not properly secreted (Araújo, 2007).

It was also found that mmy is transcriptionally regulated by Ttk. mmy tracheal expression positively depends on a mid-embryonic peak of the insect hormone 20-hydroxyecdysone. Therefore, it is proposed that Ttk and ecdysone exert opposing effects on chitin synthesis. Excess of mmy mRNA results in the abnormal deposition of the chitin filament, as occurs in ttk mutants. Defects in chitin deposition might lead to the irregular organisation of taenidia and the faint larval cuticle observed in ttk mutants. Strikingly, Ttk is also required for normal chorion production, which represents another specialised secreted layer (Araújo, 2007).

ttk mutants are defective in the formation of terminal and fusion branches. These defects are due, in part, to non-autonomous, secondary and/or pleiotropic effects of ttk. For instance, ttk mutants exhibit a dorsal closure defect, which prevents the approach and fusion of contralateral dorsal branches. Additionally, terminal and fusion branches depend on correct cell type specification, which did not reliably occur in ttk mutants. For instance, DSRF (Blistered) was missing in some presumptive terminal cells of ttk mutants, impairing terminal branch formation. These tracheal cell identity specification defects might be related to non-autonomous requirements of ttk. For instance, DSRF is not properly expressed in ttk mutants because of an abnormal expression of its regulator, Bnl (Araújo, 2007).

It is important to note that, in spite of these non-autonomous and cell fate specification defects, two pieces of evidence indicate that ttk also plays a specific and autonomous role in the formation of terminal and fusion tubes. First, markers for fusion and terminal cell specification were expressed in many tracheal cells of ttk mutants, but yet most of these cells did not form terminal or fusion branches. Second, only the tracheal expression of ttk in ttk mutants (but not the constitutive activation of the btl pathway, which regulates the terminal and fusion identity) was able to restore the formation of terminal branches (Araújo, 2007).

A common feature of terminal and fusion branches is that they both display intracellular lumina that lack detectable junctions. The cellular events that precede the formation of fusion and terminal branches differ, but the mechanisms by which their intracellular lumina form has been proposed to be comparable. It was found that, in ttk mutants, terminal and fusion cells engage in the correct cellular changes before intracellular lumen formation. However, neither of these two cell types finalised the cellular events leading to tube formation. It has been proposed that the lumen of terminal and fusion branches forms by the coalescence of intracellular vesicles that use a 'finger' tip provided by the neighbouring stalk cell as a nucleation point. Interestingly, it was found that vesicles containing luminal material are less abundant in ttk mutants. These observations suggest a new role for Ttk in the formation of intracellular lumina in distinct cell types. Intracellular lumen formation also occurs in other branched tubular structures, such as in vertebrate endothelial cells and in the excretory cell of Caenorhabditis elegans, presumably by the coalescence of vesicles. Importantly, a crucial role for vesicle formation and their fusion during intracellular tube formation has been demonstrated (Araújo, 2007).

ttk is the first gene described to be involved in intracellular lumen formation during tracheal development. Possible targets of Ttk might be genes related to the apical surface and the underlying cytoskeleton, because several of these genes are involved in C. elegans excretory canal formation. Additionally, genes involved in intracellular vesicle trafficking might also be good candidates. In this respect, several abnormalities have been detected in ttk mutants that might reflect defects in vesicle trafficking (Araújo, 2007).

Protein Interactions

Yeast and human UDP-GlcNAc-Pyp both have been shown to exhibit a UDP-N-acetylgalactosamine (UDP-GalNAc)-Pyp activity (Wang-Gillam, 2000; Peneff, 2001a). Moreover, UDP-GlcNAc can be converted to UDP-GalNAc by UDP-N-acetylglucosamine 4-epimerase (Winans, 2002; Mok, 2005). Both UDP-GlcNAc and UDP-GalNAc are essential components of the polysaccharide moieties that are added to secreted proteins upon glycosylation and GPI-anchor synthesis in the ER and the Golgi apparatus (Tonning, 2006).

In order to test whether mmy mutants display dysfunctional protein maturation along the secretory pathway, the size of the extracellular cuticle protein Knickkopf (Knk) was compared in wild-type and mmy mutant larvae on Western blots. The 689 amino acids Knk protein is predicted to be N-glycosylated at three positions (N¹⁷⁰, N³⁵⁴ and N⁶³⁶), O-glycosylated at S²⁰⁹ and T⁴⁶⁸, and to possess a GPI-anchor. In extracts of mmy^IK63 and mmy^KG08617 larvae, no wild-type sized Knk can be detected. Instead several proteins of smaller molecular weight are recognised by the Knk antiserum, indicating that post-translational modifications of Knk indeed are affected in the mutant larvae. In addition, smaller molecular weight species of the plasma-membrane protein Tout-velu (Ttv), which is predicted to be N-glycosylated at N⁷¹, N³²⁷ and N⁴⁷⁶, are present in mmy mutant larvae when compared with the wild type, whereas no size changes are observed for the intracellular membrane-bound Syntaxin1A. Treatment of protein extracts from wild-type larvae with EndoH, an enzyme that removes N-glycosylated modification from proteins, produce Knk and Ttv proteins of even higher mobility than those seen in mmy mutant larval extracts, indicating that protein glycosylation is not completely abolished in mmy mutant larvae. By contrast, in protein extracts from mmy^IL07 mutant larvae, which display strikingly similar cuticle phenotypes as knk and retroactive (rtv) mutants, and mmy^KG04349 mutant larvae, Knk and Ttv migration is not detectably altered, indicating that protein modification in these mutant backgrounds is not dramatically impaired (Tonning, 2006).

To test whether impaired modification of Knk may affect its subcellular localisation, the distribution of Knk was examined in the epidermis of stage 16 wild-type and mmy^IK63 embryos labelled with the Knk antiserum. In the wild-type embryo, Knk is detected along the apical surface of the epidermal cell, whereas in mmy^IK63 mutant embryos, only low levels of Knk signal are detected at the apical epidermal surface in addition to a faint cytoplasmic. This indicates that modification of Knk is required for its stability and localisation to the apical plasma membrane (Tonning, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

The expression of mummy was examined using a probe that detects the two transcripts. This revealed that the gene is maternally loaded; high levels of transcript are seen in freshly laid eggs, indicative of maternal expression. This ubiquitous expression declines and is reduced to a low level by stage 6. Subsequent to this, a high level of transcription occurs in the developing trachea. Expression is noticeable from stage 11 when the tracheal placodes form. This level of expression is maintained in the trachea as tracheal development proceeds, with expression continuing throughout all tracheal branches until stage 16. In mature embryos, expression was detected throughout the epidermis (Araujo, 2005).

Tracheal development begins normally in mmy mutant embryos as monitored by the anti-Trh antibody. Placodes are correctly positioned, and migration occurs normally in different directions to form the distinct tracheal branches. Thus, the initial steps of tracheal development are apparently normal until the event of tube formation. From late stage 14, the pattern of tracheal cells is not as well organized as in the wild-type. In particular, tracheal nuclei were no longer regularly spaced and do not form linear rows in the dorsal trunk. These defects worsen as development proceeds; by stage 16, the tracheal nuclei outline an irregular dorsal trunk of variable width leading to the formation of a tube with constrictions, breaks and dilations along its entire length. The tracheal cell shape was analyzed during development by detecting a UAStauGFP construct under a breathless-GAL4 promoter, a specific driver for all tracheal cells. Again, cells appeared to behave normally until the stage of tube formation when they adopted an irregular organization forming tubular structures of bubble-like appearance (Araujo, 2005).

Although Mmy is a metabolic enzyme presumably required for housekeeping functions in most cells, the specific requirements for zygotic mmy in epithelial organisation and aECM formation led to an investigation of its developmental expression pattern. In situ hybridisations to detect mmy mRNA produced a strong signal in early embryos before the start of zygotic mid-blastula transcription, indicating a maternal supply of mmy RNA. Indeed, embryos derived from mmy mutant germline clones arrest development before cellularisation, suggesting that maternal mmy provides UDP-GlcNAc for basic cellular needs during early embryogenesis. The zygotic mmy RNA is detected from stage 11, displaying a strong and temporary upregulation in different epithelial tissues. The mmy gene is first transcribed in tracheal cells just after they have invaginated as epithelial sacks from the ectoderm, and continues to be expressed at high levels in the tracheae until stage 15. This mid-embryonic mmy expression correlates with luminal chitin deposition during tube expansion (Tonning, 2005). At stage 16, mmy is strongly expressed in the epithelium of the developing salivary glands, which are not known to synthesise chitin, but may require high levels of mmy to accommodate extensive protein secretion. At late stage 16, and concurrent with chitin deposition, the mmy transcript is first detected in the epidermis (Tonning, 2006).

The dynamic and tissue-specific zygotic expression of mmy may be subjected to feedback regulation triggered by UDP-GlcNAc consumption during aECM formation. However, a hypothetical accumulation or lack of UDP-GlcNAc in kkv or mmy^IK63 mutant embryos, respectively, did not detectably alter the levels of mmy transcript, since mmy was normally expressed in these mutants (Tonning, 2006).

Requirement for chitin biosynthesis in epithelial tube morphogenesis

Many organs are composed of branched networks of epithelial tubes that transport vital fluids or gases. The proper size and shape of tubes are crucial for their transport function, but the molecular processes that govern tube size and shape are not well understood. This study shows that three genes required for tracheal tube morphogenesis in Drosophila encode proteins involved in the synthesis and accumulation of chitin, a polymer of N-acetyl-ß-D-glucosamine that serves as a scaffold in the rigid extracellular matrix of insect cuticle. In all three mutants, developing tracheal tubes bud and extend normally, but the epithelial walls of the tubes do not expand uniformly, and the resultant tubes are grossly misshapen, with constricted and distended regions all along their lengths. The genes are expressed in tracheal cells during the expansion process, and chitin accumulates in the lumen of tubes, forming an expanding cylinder that is proposed to coordinate the behavior of the surrounding tracheal cells and stabilize the expanding epithelium. These findings show that chitin regulates epithelial tube morphogenesis, in addition to its classical role protecting mature epithelia (Devine, 2005).

The extant mutation (k13717) in the cystic tube expansion gene is a recessive mutation that dramatically alters tracheal tube diameter and shape in homozygous embryos. cystic^k13717 is a P[lacZ]-induced mutation, but the identified transposon insertion is unlinked to the cystic phenotype. To facilitate genetic analysis and mapping of cystic, 4,394 ethyl methanesulfonate-mutagenized chromosomes were screened and 20 recessive alleles were identified that failed to complement the lethality of cystic^k13717. Fifteen mutations (LM1, LM16, LM23, LM24, LM29, LM33, LM35, LM42, LM44, LM45, LM47, LM49, LM51, LM55, and LM59) severely disrupted tracheal tube morphology, showing constrictions and cyst-like expansions along the tracheal tubes in homozygous embryos, similar to the original cystic allele (Class III alleles). Class II homozygotes (LM21 and LM32) displayed mild tracheal defects, with slight constrictions in the tracheal lumen. Class I homozygotes (LM7, LM15, and LM19) had normal tracheal morphology. Transheterozygous mutant combinations (LM42/LM1, LM 51/k13717, k13717/LM21, LM16/LM21, and LM16/LM15) displayed phenotypes similar to, or intermediate between, those of the corresponding homozygotes. The tracheal phenotypes of LM1, LM24, and LM42 hemizygous embryos [mutation in trans to cystic deficiency Df(2L)BSC6] were not more severe than those of the corresponding homozygotes, indicating that Class III alleles are null (amorphic) or strong loss-of-function mutations. General embryonic morphology and gut morphology appeared normal under differential interference contrast microscopy, except for an impenetrant dorsal closure defect observed in Class III homozygotes. No defects were detected in heterozygous embryos, indicating that the mutations are fully recessive (Devine, 2005).

Early events in tracheal development, including tracheal sac formation and primary branch budding and outgrowth, appear normal in Class III cystic mutants. Defects were detected at late stage 14 (~11 h after egg lay), when the dorsal trunk begins to dilate. Imaging of tube expansion in live embryos shows that in wild type, the dorsal trunk lumen gradually and uniformly dilates to three times its original diameter over a several-hour period. In Class III cystic mutants, expansion occurs unevenly. Portions of the tube dilated poorly, whereas other regions expanded too much. As expansion proceeded, the dorsal trunk becomes progressively more misshapen, with constricted and distended regions all along its length (Devine, 2005).

Expression of the 2A12 and AS55 luminal markers, which normally begin to be expressed just before tube expansion, is also severely compromised in cystic mutants. In wild type, 2A12 antigen first appears in the tracheal epithelium at stage 14 in cytoplasmic punctae or vesicles and then accumulates along the apical (luminal) surface during stage 15 (~12 h after egg lay) as tubes expand. Although the punctate vesicular pattern was observed at stages 14 and early 15 in all cystic mutants examined, in strong alleles, 2A12 antigen accumulation at the luminal surface was markedly reduced (Class IIIa alleles) or absent (Class IIIb alleles). Likewise, AS55 antigen, which normally begins accumulating at the lumen surface at stage 14, was severely reduced or absent in Class III alleles. By contrast, expression levels of epithelial polarity markers including apicolateral Crumbs and E-cadherin, basolateral Na⁺-K⁺ ATPase, and septate junction proteins Neurexin IV, Coracle, and Fasciclin III appear normal in cystic mutants (Devine, 2005).

cystic^LM1 was mapped to a 300-kb region on chromosome II by meiotic recombination. Deficiency mapping further localized cystic^LM1 to an ~8-kb interval that overlaps the location of a 61-kb deletion adjacent to the P[lacZ] insertion on the cystic⁰³⁸⁵¹ chomosome, a P element-induced allele of cystic. The interval contained two candidate genes, CG9531 and CG9535. Sequencing of their coding regions in cystic alleles identified deletion, nonsense, or missense mutations in CG9535 in all 15 alleles analyzed, and the severity of the molecular defect corresponded to the strength of the tracheal phenotype. No changes were found in the coding region of the other candidate gene in the cystic alleles analyzed (LM1, LM16, and LM42), and only two insignificant nucleotide changes were found among ~10⁵ nucleotides sequenced or surveyed by temperature gradient gel electrophoresis in the five genes neighboring the candidate interval for the 17 cystic alleles analyzed. It is concluded that CG9535 is cystic (Devine, 2005).

cystic encodes the Drosophila homolog of UDP-N-acetylglucosamine diphosphorylase (EC 2.7.7.23), which catalyzes formation of UDP-N-acetylglucosamine and is the penultimate enzyme in the chitin synthesis pathway. The enzyme is highly conserved in eukaryotes: Cystic is 51% identical to human UAP1/AGX1. The structure and enzymology of this family of enzymes have been extensively studied, and residues important for substrate binding and catalysis are conserved in Cystic. The three missense mutations (LM16, LM24, and LM55) in the diphosphorylase consensus motif located in the substrate-binding pocket and the deletion and frameshift mutations (LM33, LM47, LM49, LM51, 03851, and k13717) all have a strong tracheal phenotype, consistent with their assignment as null alleles. The other strong alleles (LM1, LM35, and LM42) alter other conserved residues in the substrate-binding site. cystic is expressed in the developing tracheal system beginning at late stage 12/early 13 and continuing through stages 14 and 15 when tube expansion occurs (Devine, 2005).

gnarled is another tracheal tube expansion gene with a phenotype similar to strong cystic mutants. gnarled maps to cytologic interval 85E11-F16, the same region as knickkopf (knk), a gene implicated in epidermal cuticle development. knk¹ mutants display a tracheal tube expansion defect indistinguishable from that of gnarled^ex59, and knk¹ fails to complement gnarled^ex59 for tracheal phenotype and viability. Expression of a UAS-knk transgene in the developing tracheal system under btl-GAL4 control rescues the tracheal defects and restores viability to both gnarled^ex59 and knk¹ mutants. It is concluded that gnarled and knk are the same gene (Devine, 2005).

knk RNA is expressed in the developing tracheal system in the same pattern as cystic. knk encodes a novel protein. To investigate its cellular localization, a genomic rescue construct was generated with the coding sequence for GFP inserted at the 3' end of the knk coding region. The Knk-GFP fusion gene was expressed in a similar pattern to the endogenous gene and rescued knk¹ to viability. Knk-GFP localizes diffusely toward the apical (luminal) surface of tracheal cells, where chitin is deposited. There were also discrete punctae of Knk-GFP at the apical surface or just within the tracheal lumen that colocalized with 2A12 punctae. Although knk is required for chitin accumulation in the lumen, Knk-GFP protein localized near but did not overlap the distribution of chitin. This suggests a role for Knk in targeting or secretion of chitin or chitin synthesis enzymes (Devine, 2005).

krotzkopf verkehrt (kkv) encodes a chitin synthase homolog that, like knk, is required for epidermal cuticle development. kkv is expressed in the developing tracheal system, and kkv mutants have a tracheal tube expansion defect similar to knk and cystic. Expression of a UAS-kkv transgene in the developing tracheal system ameliorates the kkv tube expansion defect (Devine, 2005).

The expression of chitin synthesis genes in developing tracheal cells suggested that chitin might be produced during tube expansion. Indeed, probing wild-type embryos with a rhodamine-conjugated chitin-binding protein revealed chitin in the tracheal lumen at stage 13, just before tube expansion begins. Chitin continued to accumulate throughout the period of tube expansion. Confocal optical sectioning showed that chitin was not restricted to the apical epithelial surface as it is in mature tracheal tubes and other epithelia. Rather, it formed an expanding cylinder that filled almost the entire luminal space. No chitin was detected in the developing tracheal system of cystic and kkv mutants. Some chitin was detected in knk mutants at the onset of tube expansion but not later in the process. Thus, chitin is synthesized and forms an expanding cylinder in the lumen of developing tracheal tubes, and this process is disrupted in the expansion mutants (Devine, 2005).

Analysis of tracheal tubes during the expansion period by transmission electron microscopy (TEM) did not detect the luminal chitin cylinder, presumably because this dynamic form of chitin is not preserved under the TEM fixation and staining conditions. However, the TEM analysis did reveal the presence of a thin extracellular matrix lining the apical (luminal) surface of the tracheal epithelium before expansion begins. In newly expanded tubes, small ridges formed in the matrix. These later become taenidia, the circumferential ridges in tracheal cuticle that contain a procuticle chitin core. In Class III cystic mutants (LM1 and LM42), the thin matrix formed but the ridges did not, and the mature tracheal epithelium had a thin epicuticle but virtually no procuticle or taenidia. These results imply that there is a second, more conventional, form of chitin, stable to TEM fixation and staining, which begins to accumulate during or just after tube expansion and forms the taenidial rings of tracheal cuticle. No ultrastructural defects were detected in epidermal cuticle in the cystic mutants, and only subtle defects were detected in epidermal cuticle in whole-mount cuticle preparations; this suggests that other gene(s) provide cystic function in the epidermis, or there is a small maternal contribution of cystic that is sufficient for epidermal but not tracheal function of the gene (Devine, 2005).

Chitin is one of the most abundant biopolymers in nature. It has long been known to serve as a scaffold for the stiff cuticle that lines and protects exposed epithelia, including epidermal and tracheal epithelia. These results provide strong evidence that chitin also functions in epithelial morphogenesis. Chitin synthesis genes are expressed during tracheal development, and chitin accumulates throughout the lumen of tracheal tubes, forming an expanding cylinder whose growth parallels that of the surrounding epithelium. In mutants lacking chitin, expansion is uncoordinated, with some regions of the epithelium dilating poorly while others overexpand, forming grossly misshapen tubes with constrictions and dilatations all along their length. Although these results strongly implicate chitin in tracheal morphogenesis, the data do not exclude the possibility that the chitin synthesis genes have functions other than their roles in chitin synthesis that could cause or contribute to the tracheal phenotype (Devine, 2005).

It is proposed that the expanding chitin cylinder in the luminal matrix coordinates the behavior of the surrounding tracheal cells and stabilizes the expanding epithelium. This ensures that tracheal tubes expand progressively and uniformly. As tubes reach the appropriate size, a developmental transition must occur that arrests expansion and alters chitin structure or synthesis to form taenidial rings and the rigid cuticle that protects the apical surface of the mature tracheal epithelium. This transition might involve a change in the chitin secretion mechanism or in its assembly into fibrils or its association with matrix proteins. Ultimately, the central chitin cylinder must be degraded or remodeled and cleared along with other luminal contents as the tubes fill with gas and become functional for respiration several hours later. To test the proposed roles of chitin in the model, it will be necessary to find ways to selectively alter or abolish the different forms of chitin (Devine, 2005).

Although chitin is rare in vertebrates, many vertebrate tubes contain related carbohydrates or uncharacterized fibrillar material at their apical surface or throughout the lumen. Although it is not known whether these substances function in tube morphogenesis, there is evidence that at least some of these molecules, including chondroitin sulfate and hyaluronic acid, can influence related morphogenesis processes such as epithelial invagination. There is also a long history in the field of tissue engineering of using cylinders ('mandrils') made of synthetic and/or biological materials as templates for constructing and shaping blood vessels for clinical use. It will be important to investigate the structure and function of the luminal contents of developing vertebrate tubes to determine whether they contain cylinders like chitin and engineered mandrils that coordinate the morphogenesis of the surrounding epithelium (Devine, 2005).

EFFECTS OF MUTATION

To identify new genes involved in tracheal morphogenesis, a collection of EMS-induced mutants was screened using the 2A12 antiserum that recognizes an epitope at the lumen of the tracheal tubes. Among the new mutants found, one (H053) did not display a continuous luminal structure and instead showed a very dispersed tracheal staining. Upon examination of these mutant embryos with an antibody recognizing the trachealess (trh) gene product, it was confirmed that the tracheal cells in these embryos were present and were reasonably positioned in a tubular manner similar to the wild-type. However, after more detailed analysis, the arrangement of the cells in H053 was found to be rather distorted, lacking their typical aligned structure (Araujo, 2005).

In an independent screen for mutants showing CNS phenotypes, two mutants (GA74 and GA760) were identified with a subtle CNS phenotype where the commissural pathways appear diffuse and there is a reduction in the thickness of the longitudinal tracts. The fasciclin-II-positive fascicles within these embryos are not as well defined as in wild-type, and fasciclin-II-positive axons are observed extending towards the midline. These phenotypes are characteristic of defects in midline signaling (Araujo, 2005).

Further examination of the mutant phenotypes showed that both H053 and GA74 mutants displayed simultaneously the tracheal and CNS phenotype. Both mutants were uncovered by the same deficiencies, and a complementation analysis showed that they failed to complement one another, both for lethality and for the embryonic phenotypes. Therefore, H053 and GA74 define a single complementation group. During the phenotypic analysis of these mutants, it was realized that their tracheal defect is reminiscent of that previously described for the cystic (cyst) mutant (Beitel, 2000). It was also observed that H053, GA74 and cyst display a cuticle phenotype similar to that seen in mummy (mmy) mutants (Nüsslein-Volhard, 1984). In addition, it was found that the cyst and mmy mutations are also uncovered by the same deficiencies, and all these independently induced mutants failed to complement each other; therefore, they are alleles of the same gene. Since mummy (mmy) is the name of the earliest induced mutant, this name has been retained for all the alleles (Araujo, 2005).

To identify the genomic location of the mmy gene affected in the tracheal and CNS mutants, two alleles, mmy^GA74 and mmy^GA760, were mapped by performing a lethal complementation analysis with a set of deficiencies from the Bloomington Stock Center. This revealed that Df(2L)BSC6, which deletes the region 26D3-E1 to 26F4-7, fails to complement the mmy alleles. An overlapping deficiency from the DrosDel collection that removes 26C1 to 26D7, Df(2L)ED380, complemented the alleles as did a neighboring Bloomington deficiency, Df(2L)BSC7, that deletes the interval from 26D10-E1 to 27C1. This revealed that the mmy gene lies in the region 26D7-26E1. A series of lethal P-element insertions in this region were tested, and the insertion KG08617 failed to complement all mmy alleles. Transheterozygote combinations of mmy^GA74 with KG08617 displayed the same phenotypes as the mmy^GA74 homozygotes, suggesting that the gene disrupted by KG08617 is mmy. KG08617 is inserted within an intron of the predicted gene CG9535, suggesting that CG9535 is the gene responsible for the mmy phenotype. To confirm this, the P-element was excised precisely which resulted in reversion to wild-type. To further confirm that CG9535 is affected in the mutants, the mmy^GA74 and mmy^H053 chromosomes were sequenced to reveal changes of Val419Asp and Trp199STOP, respectively (Araujo, 2005).

mummy encodes an enzyme required for chitin and glycan synthesis, involved in trachea and embryonic cuticle development

The original analysis of the mmy tracheal phenotype using the 2A12 antibody showed a discontinuous lumenal structure. To further investigate this, the localization of the Piopio protein was analyzed. In wild-type embryos, Pio accumulates in the tracheal lumen where it is thought to be part of the extracellular matrix that provides a structural network in the luminal space (Jazwinska, 2003). Pio accumulation appears normal in the mmy trachea, but, at stage 14, when wild-type embryos already show a continuous lumen, the mmy lumen displays interruptions between metameres, suggesting a lack of a continuous luminal structure or a delay in the fusion process. Noticeably, the general accumulation of Pio seems to be unaffected in mmy mutants, as opposed to the accumulation of the lumen epitope recognized by the 2A12 antibody, suggesting that different luminal structures present in the tracheal tubes have different requirements for localization. At stage 16, Pio accumulation is still lumenal, highlighting the bubble-like appearance of the dorsal trunk (Araujo, 2005).

How can a UDP-GlcNAc diphosphorylase affect tracheal morphogenesis? This enzyme plays a key role in the metabolic pathway leading to chitin formation, a component of the tracheal cuticle and a main component of the arthropod exoskeleton. UDP-GlcNAc is the major constituent of chitin and the substrate for chitin synthase (Merzendorfer, 2003). In the embryo, chitin can be visualized by the incorporation of Fluostain (Moussian, 2005a), a chitin-adsorbing brightener, and terminal GlcNAc residues can be detected by wheat germ agglutinin (WGA; Wright, 1984), a lectin (Araujo, 2005).

mmy embryos were stained with Fluostain and it was found that chitin was not present in the trachea of these mutants, suggesting that this could be the cause of their tracheal phenotype. In addition, WGA incorporation in mmy embryos was practically absent, revealing the lack of terminal GlcNAc residues at the cell surface. In wild-type embryos, WGA stained both apical and basal cell surfaces and accumulated in the tracheal lumen, cuticle and gut. In mature mmy embryos, however, only a very faint staining could be detected at the surface of cells (Araujo, 2005).

Previous reports indicated that the tracheal epithelium of mmy^cyst mutants is permeable as assessed by a dye permeability assay, suggesting a loss of the trans-epithelial diffusion barrier in this mutant (Paul, 2003). This is a relevant point since septate junctions (SJs), which have a prominent role in the establishment of this diffusion barrier, have also been shown to be critical for the control of tracheal tube size. For this reason, whether SJs could be affected in mmy mutants was tested. Contrary to what was observed in mutants for SJ components, it was found that upon dye injection the diffusion barrier was not completely abolished in the mmy trachea. Rather, some independent tracks of the tube remained impermeable, while others showed a loss of the diffusion barrier as judged by dye uptake. These results suggested that the general diffusion barrier mechanism mediated by the SJs was not wholly impaired in mmy mutants. On the contrary, breaks and discontinuities within the lumen of the tube could be the origin of the permeabilization defects (Araujo, 2005).

Supporting the permeabilization results, it was observed that, even at late embryonic stages, when the tube is severely affected, components of the SJs such as Coracle (Cora) and Discs-large (Dlg) maintain a correct localization both in the tracheal tubes, epidermis and salivary glands. In addition, Stranded-at-second (Sas), a marker for the apical surface of cells, showed a correct localization in mmy. Taken together, these results indicate that the apical/basal polarity of the mutant cells is maintained and that the SJ components are not mislocalized in mmy embryos (Araujo, 2005).

By marking the cytoplasm of the tracheal cells with GFP, it was found that, despite the bubble-like appearance of the dorsal trunk, its cells still display structural continuity. But, even though there is a continuity between the cells of the dorsal trunk, its lumen forms separate bubble-like structures. The peculiar morphology of the mmy mutant trachea suggests that the observed phenotype could be due to incorrect lumenal fusion and expansion between the independent tubular structures from adjacent metameres. An important step in this process is the formation of three cadherin rings at the fusion point. Complete failure of fusion, due to mutations in E-cadherin, leads to the formation of separate unfused structures in each metamere. In spite of detecting the presence of E-cadherin on the surface of cells in mmy embryos, the formation of the cadherin rings in their dorsal trunk could not be observed, suggesting that the correct fusion of the two adjacent lumens into a continuous tube is not fully achieved. These cadherin rings are the result of the fusion process and, at late embryonic stages, mark the site where the lumen connections occur after fusion of the dorsal trunk cells. In order to check for the presence of fusion cells in mmy embryos, the expression was analyzed of two markers that are characteristic of these cell types, Fusion-3 and Headcase. Fusion-3 is expressed in all fusion cells from stage 13/14, and Headcase is expressed in all but the dorsal trunk fusion cells from stage 15. Both markers were detected in mmy as in the wild-type, indicating that fusion cells appear to be correctly specified in mmy mutants, although fusion is not completely accomplished (Araujo, 2005).

All the above results suggest that the absence of chitin could be the main cause of the abnormal shape of the mmy tracheal system. To corroborate this hypothesis and to examine tube architecture in mmy embryos, transmission electron microscopy (TEM) analysis was performed of the tracheal system of mature mmy embryos. Three main features were recognized at the ultrastructural level that diverge from the wild-type organization of the tracheal lumen. (1) The taenidial folds inside the tubes are not properly formed. The taenidial folds are annular rings around the tracheal lumen that are thought to be important to give some stiffness to the tubes and to allow them to expand and contract along their length. These taenidia, which become visible as cuticular protrusions in transverse and longitudinal sections, are not present in the tracheal lumen of mmy embryos. (2) In contrast to what occurs in the wild-type, the luminal structures are not in close apposition to the membranes of the surrounding tracheal cells; instead, the lining of the tube appears detached. (3) In mmy embryos, at the apical membranes of the tracheal cells, a higher number of microvillae were detected than in same stage wild-type embryos. Microvillae are detected during the first steps of envelope formation and are thought to have an active role in the formation of cuticular structures. In mmy mutants, perhaps due to incorrect synthesis of cuticular luminal components, these microvillae continue to be detected at later stages than in wild-type embryos. As a consequence, the apical side of the epidermal cells does not form a continuous structure with the lumen (Araujo, 2005).

Taken together, these observations led to the conclusion that the tracheal lumen of mmy embryos is missing the characteristic luminal architecture present in the wild-type trachea and that this can be related to the lack of the cuticular structures due to chitin absence (Araujo, 2005).

To assess the requirement of chitin in the proper organization of the tracheal system, whether similar mutant phenotypes were found in other mutants that affect chitin synthesis was analyzed. Recently, it has been shown that the krotzkopf verkehrt (kkv) gene encodes a chitin synthase enzyme (CS-1); as a result, there is no chitin in the trachea of kkv embryos (Ostrowski, 2002: Moussian, 2005a). Since both the Mmy and Kkv enzymes are involved in the later stages of the chitin synthesis pathway, the analysis of the kkv mutant trachea was extended and compared to mmy (Araujo, 2005).

kkv tracheal tubes are similar to the tubes in mmy mutant embryos; they display comparable constrictions and dilations. However, the overall tube structure in kkv is not as distorted. Specifically, the dorsal trunk lumen of kkv mutants is continuous and does not show the fragmented and separated bubble-like structures present in the mmy mutants. This correlates with the observation that the cadherin rings produced by the fusion cells are present in the kkv mutant dorsal trunk, suggesting that the fusion process allows the formation of a continuous dorsal trunk lumen. A dye permeability assay was performed to test the trans-epithelial diffusion barrier in kkv mutants. As in wild-type, the rhodamine-labeled dextran did not show any diffusion into the tracheal lumen, indicating that the diffusion barrier mechanism mediated by the SJs is not impaired in kkv mutants (Araujo, 2005).

In addition, no CNS phenotypes were detected in kkv embryos. The CNS of kkv mutants is apparently normal, which supports the hypothesis that the CNS defects observed in mmy are not attributable exclusively to the lack of chitin (Araujo, 2005).

These observations suggest that there are, most likely, some chitin-independent phenotypes associated with the mmy mutants. WGA staining is abolished in the tracheal cells of mature mmy embryos, but present in those of kkv mutants, suggesting that the absence of other products detectable by this lectin can be responsible for the more severe tracheal phenotype of mmy mutants when compared to kkv (Araujo, 2005).

Chitin is a component of the insect embryonic cuticle with a major role in its formation and assembly. The role of chitin in the Drosophila embryonic cuticle has recently been extensively analyzed (Moussian, 2005a). It was shown that the embryonic cuticle of kkv embryos detaches from the body and loses its integrity but keeps some cuticular structures like the head skeleton, the denticle belts and the hairs. Cuticle preparations of mmy mutant embryos were examined, and it was found that these cuticles are much more disrupted than those reported for kkv embryos (Moussian, 2005a). No specific structures were recognized, and there were no signs of denticle belts or hairs; in some cases, what could be remnants of the head skeleton and sclerotic fragments were detected. Indeed, when mmy mutant embryos were devitelinized, no cuticle preparation could be recovered (Araujo, 2005).

As in the trachea, both chitin and other GlcNAc containing products, as detected by WGA, are present in the wild-type embryonic cuticle but absent in mmy. However, while kkv embryonic cuticle lacks chitin, it still contains terminal GlcNAc residues as detected by WGA incorporation. Again, as in the tracheal system, this difference could explain the more dramatic effects on the embryonic cuticle of mmy embryos (Araujo, 2005).

To examine in more detail the defects in mmy embryonic cuticle, its structure was analyzed by TEM. As described previously (Locke, 2001), in the wild-type cuticle of mature embryos, it was possible to distinguish the envelope, the epicuticle and the procuticle; below these, an adhesion zone is thought to mediate the adhesion with the epidermal cells. Among these layers, the procuticle is composed of stacks of chitin laminae oriented parallel to one another in a highly organized structure. In mmy mature embryos, only the envelope and the epicuticle, the chitin-free layers, seem to maintain remainders of their structure. In particular, there is no sign of the highly organized structure of the procuticle, and the underlying epithelial cells appear detached in many places, indicating an overall disruption of the cuticle and its interaction with the epidermis (Araujo, 2005).

Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila

Many epithelia produce apical extracellular matrices (aECM) that are crucial for organ morphogenesis or physiology. Apical ECM formation relies on coordinated synthesis and modification of constituting components, to enable their subcellular targeting and extracellular assembly into functional matrices. The exoskeleton of Drosophila, the cuticle, is a stratified aECM containing ordered chitin polysaccharide lamellae and proteinaceous layers, and is suited for studies of molecular functions needed for aECM assembly. Drosophila mummy (mmy) mutants display defects in epithelial organisation in conjunction with aberrant deposition of the cuticle and an apical matrix needed for tracheal tubulogenesis. mmy encodes the UDP-N-acetylglucosamine pyrophosphorylase, which catalyses the production of UDP-N-acetylglucosamine, an obligate substrate for chitin synthases as well as for protein glycosylation and GPI-anchor formation. Consequently, in mmy mutants GlcNAc-groups including chitin are severely reduced and modification and subcellular localisation of proteins designated for extracellular space is defective. Moreover, mmy expression is selectively upregulated in epithelia at the time they actively deposit aECM, and is altered by the moulting hormone 20-Hydroxyecdysone, suggesting that mmy is part of a developmental genetic program to promote aECM formation (Tonning, 2006).

Since the chitin containing procuticle is affected in both strong and weak mmy mutant larvae, the presence of chitin was tested using gold labelling with the lectin wheat germ agglutinin (WGA). WGA binds GlcNAc-groups on glycosylated proteins, glycosaminoglycans as well as chitin, but on specimens embedded in Epon and sectioned for TEM, WGA recognises only chitin. Although chitin is present in wild-type and in the weak mmy^IL07 mutant procuticle, no chitin is detected in mutant larvae homozygous for the strong mmy^IK63 allele, arguing that mmy is essential for chitin synthesis. However, mmy appears to have additional roles in cuticle differentiation, since loss of chitin causes a compensatory increase in cuticular protein deposition (Moussian, 2005a), which is not detected in mmy^IK63 mutants (Tonning, 2006).

TEM analysis of the mmy^IK63 cuticle also revealed irregular cell shapes in the underlying epidermis. These cells are cuboidal, rather than flattened like the wild-type epidermal cells, and their lateral membranes are not undulated. Epithelial cell shape is determined by the cytoskeleton and cell-cell contacts. Using high resolution TEM analysis, it was found that the orientation of microtubules in wild-type, mmy^IL07 and mmy^IK63 mutant larval epidermal cells are indistinguishable, and that the adherens and septate junctions (AJ and SJ) are positioned normally along the lateral membrane. However, in the mmy^IK63, but not mmy^IL07 mutant embryos, spacing between the epidermal cells at the AJ appears wider than in the wild type, and the characteristic ladder-like structure of the SJs is missing. SJ integrity was examined in the two mmy mutant epithelia, by labelling for the two SJ components Fasciclin 3 (Fas3) and Discs large 1 (Dlg1). Both SJ proteins are normally distributed in the weak mmy^IL07 epithelia, and the intracellular Dlg1 protein is also normally localised in the more severe mmy^IK63 mutants. By contrast, the transmembrane Fas3 is mislocalised along the lateral membrane in the columnar epithelia of the hindgut and salivary gland in some mmy^IK63 embryos. Thus, the disrupted SJ ladder in mmy^IK63 mutants may reflect a requirement for Mmy in the correct localisation of membrane-bound SJ components. Since chitin-deficient embryos have no detectable defects in SJ assembly or function (Moussian, 2005a), and chitin-deposition appears unaffected in embryos with disrupted SJ components (Tonning, 2005), these results point to two parallel requirements for mmy in chitin synthesis and maturation of cell-cell contacts (SJs) (Tonning, 2006).

The tracheal (respiratory) tubular network is an epithelium that undergoes extensive cell rearrangements during embryonic development, and relies on a luminal (apical) matrix for uniform tube growth. Since chitin is an essential component of this luminal matrix, tests were performed to see whether mmy mutant embryos, which lack cuticular chitin, also display tracheal tube size defects. The wild-type developing tracheal system can be visualised with the lumen specific antibody 2A12. The same staining reveals a perfectly patterned tracheal system in embryos homozygous for the weak mmy^IL07 allele, although the dorsal trunk (DT) lumen diameter is slightly irregular at stage 15 and becomes excessively elongated at stage 16. The tracheal mmy^IK63 mutant lumen, however, fails to label with the 2A12-antibody, although the typical intracellular vesicular 2A12-staining is present. Tracheal tube shapes in mmy^IK63 mutants were therefore visualised with the pan-tracheal LacZ-enhancer-trap 1-eve-1. Detection of the 1-eve-1 product shows that mmy^IK63 mutants also display normal tracheal branch patterning, but tube diameter is severely defective. The mmy^IK63 DT fusion branch lumens fail to expand and remain constricted, whereas other parts of the DT trunk become excessively dilated; during stage 16, these DTs develop huge cyst-like structures. Thus, mmy is indeed required for uniform tube expansion (Tonning, 2006).

The luminal tracheal chitin matrix can be detected both with a FITC-conjugated chitin-binding probe (CBP) and WGA (Tonning, 2005). CBP appears specific for chitin and labels a broad filamentous chitin cable within the wild-type tracheal lumen that is absent in krotzkopf verkehrt (kkv) mutant embryos defective in chitin synthase (Tonning, 2005). In mutants for the weak mmy^IL07 allele, CBP labels a luminal cable with similar intensity to the wild type, but its filamentous appearance is slightly perturbed, implying that the chitin matrix is not properly assembled. By contrast, embryos homozygous for the strong mmy^IK63 allele completely fail to label with CBP, indicating a need for mmy also in tracheal chitin synthesis. WGA labelling of wild-type and mmy^IK63 mutant embryos was compared. In the wild-type tracheae, WGA labels not only the broad luminal chitin cable, but also the luminal surface that probably represents glycosylated proteins. In the mmy^IK63 mutant tracheae, both the luminal and apical WGA-staining is severely reduced. This is different from chitin-deficient embryos, where WGA detects GlcNAc-groups along the apical cell surface and within the lumen, although the broad luminal cable is absent, arguing that mmy function is required not only for chitin synthesis, but also for other GlcNAc-containing components (Tonning, 2006).

Despite the severe tracheal tube defects in mmy^IK63 mutants, it was found that apicobasal polarity appears normal. The transmembrane protein Crumbs (Crb), which is required for cell polarisation and assembly of the adherens junctions (AJs), localises to the apical tracheal membrane in both wild-type and mmy^IK63 mutant embryos, and the microtubule minus end reporter, Nod:ß-gal, appears to accumulate correctly in the apical tracheal cell domain in the mutant. Furthermore, no significant defects in Fas3 localisation were observed in the mmy^IK63 mutant tracheal epithelium. Apical secretion of the luminal protein Piopio (Pio), which is required to form narrow tubes with autocellular junctions (Jazwinska, 2003), was normal in mmy^IK63 mutants. This indicates that zygotic mmy is not generally required for apical protein secretion (Tonning, 2006).

mmy^IK63 mutants display severely reduced WGA-labelling in embryonic tissues not known to produce chitin, including the early epidermis and the salivary glands. Moreover, labelling for Crb revealed local tube dilations in the single-cell-layered Malphigian tubules (both in the common ureter and in the individual tubules), a failure of the salivary gland lumens to enlarge in late embryogenesis, and a dorsal open phenotype in ~10 % of the mmy mutant embryos, which may reflect a need for high levels of GlcNAc-containing substances for these processes (Tonning, 2006).

Since mmy belongs to the group of Halloween mutants, including shadow (sad) and shade (shd), which disrupt enzymes needed for biosynthesis of the insect hormone 20-hydroxyecdysone, it was asked whether temporal expression of mmy in different epithelia may depend on 20-hydroxyecdysone. The sad gene encodes a mitochondrial P450 enzyme (CYP315A1), a C2-hydroxylase that catalyses the formation of ecdysone from 2-deoxyecdysone; shd encodes the enzyme CYP314A1, which is responsible for the mono-oxygenation of ecdysone to generate the active 20-hydroxyecdysone. Indeed, two aspects of mmy expression are similarly altered in embryos mutant for sad and shd. (1) It was found that shd and sad mutants lack the mid-embryonic up-regulation of tracheal mmy expression, and (2) mmy expression is prematurely upregulated in the epidermis, salivary gland and proventriculus. Thus, a mid-embryonic 'ecdysone pulse' appears essential to control the temporal expression of mmy in different embryonic epithelia (Tonning, 2006).

REFERENCES

Search PubMed for articles about Drosophila Mummy

Araujo, S. J., Aslam, H., Tear, G. and Casanova, J. (2005). mummy/cystic encodes an enzyme required for chitin and glycan synthesis, involved in trachea, embryonic cuticle and CNS development--analysis of its role in Drosophila tracheal morphogenesis. Dev. Biol. 288(1): 179-93. 16277981

Araújo, S. J., Cela, C. and Llimargas, M. (2007). Tramtrack regulates different morphogenetic events during Drosophila tracheal development. Development 134(20): 3665-76. PubMed citation: 17881489

Beitel, G. J. and Krasnow, M. A. (2000). Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127: 3271-3282. 10887083

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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

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Gilbert, L. I. (2004). Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster. Mol. Cell Endocrinol. 215: 1-10. 15026169

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Jazwinska, A., Ribeiro, C. and Affolter, M. (2003). Epithelial tube morphogenesis during Drosophila tracheal development requires Piopio, a luminal ZP protein. Nat. Cell Biol. 5: 895-901. 12973360

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Moussian, B., Schwarz, H., Bartoszewski, S. and Nüsslein-Volhard, C. (2005a). Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster. J. Morphol. 264: 117-130. 15747378

Moussian, B., Söding, J., Schwarz, H. and Nüsslein-Volhard, C. (2005b). Retroactive, a membrane-anchored extracellular protein related to vertebrate snake neurotoxin-like proteins, is required for cuticle organization in the larva of Drosophila melanogaster. Dev. Dyn. 233: 1056-1063. 15844167

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Paul, S. M., et al. (2003). The Na/K ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system. Development 130: 4963-4974. 12930776

Peneff, C., Ferrari, P., Charrier, V., Taburet, Y., Monnier, C., Zamboni, V., Winter, J., Harnois, M., Fassy, F. and Bourne, Y. (2001a). Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture. EMBO J. 20: 6191-6202. 11707391

Peneff. C., Mengin-Lecreulx, D. and Bourne, Y. (2001b). The crystal structures of Apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase. J. Biol. Chem. 276: 16328-16334. 11278591

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: 423-430. 16139230

Tonning, A., Helms, S., Schwarz, H., Uv, A. E. and Moussian, B. (2006). Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila. Development 133(2): 331-41. 16368930

Wang-Gillam, A., Pastuszak, I., Stewart, M., Drake, R. R. and Elbein, A. D. (2000). Identification and modification of the uridine-binding site of the UDP-GalNAc (GlcNAc) pyrophosphorylase. J. Biol. Chem. 275: 1433-1438. 10625695

Winans, K. A. and Bertozzi, C. R. (2002). An inhibitor of the human UDP-GlcNAc 4-epimerase identified from a uridine-based library: a strategy to inhibit O-linked glycosylation. Chem. Biol. 9: 113-129. 11841944

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Wu, V. M. and Beitel, G. J. (2004). A junctional problem of apical proportions: epithelial tube-size control by septate junctions in the Drosophila tracheal system. Curr. Opin. Cell Biol. 16: 493-499. 15363798

date revised: 20 April 2007

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