The Interactive Fly

Genes involved in tissue and organ development

Ectoderm and Epidermis

What distinguishes ectoderm from epidermis?

Genes expressed in the amnioserosa

Dysfunction of Oskyddad causes Harlequin-type ichthyosis-like defects in Drosophila melanogaster

A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea

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

Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development

Genes expressed in ectoderm and epidermis

*** indicates a special link to ectoderm specific information

What distinguishes ectoderm from epidermis?

The ectoderm is the outer germ layer of the embryo, to be distinguished from the endoderm and mesoderm. Epidermis, derived from ectoderm, is the outer epithelial layer of the embryo, larva and adult; it secretes cuticle, the exoskeleton of the fly.

genes expressed in ectoderm

Dysfunction of Oskyddad causes Harlequin-type ichthyosis-like defects in Drosophila melanogaster

Prevention of desiccation is a constant challenge for terrestrial organisms. Land insects have an extracellular coat, the cuticle, that plays a major role in protection against exaggerated water loss. This study reports that the ABC transporter Oskyddad (Osy)-a human ABCA12 paralog-contributes to the waterproof barrier function of the cuticle in the fruit fly Drosophila melanogaster. The reduction or elimination of Osy function provokes rapid desiccation. Osy is also involved in defining the inward barrier against xenobiotics penetration. Consistently, the amounts of cuticular hydrocarbons that are involved in cuticle impermeability decrease markedly when Osy activity is reduced. GFP-tagged Osy localises to membrane nano-protrusions within the cuticle, likely pore canals. This suggests that Osy is mediating the transport of cuticular hydrocarbons (CHC) through the pore canals to the cuticle surface. The envelope, which is the outermost cuticle layer constituting the main barrier, is unaffected in osy mutant larvae. This contrasts with the function of Snu, another ABC transporter needed for the construction of the cuticular inward and outward barriers, that nevertheless is implicated in CHC deposition. Hence, Osy and Snu have overlapping and independent roles to establish cuticular resistance against transpiration and xenobiotic penetration. The osy deficient phenotype parallels the phenotype of Harlequin ichthyosis caused by mutations in the human abca12 gene. Thus, it seems that the cellular and molecular mechanisms of lipid barrier assembly in the skin are conserved during evolution (Wang, 2020).

A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea

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 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. The mmy mutant phenotype is similar to that of the so-called 'Halloween' mutants, which fail to produce the differentiation hormone 20-Hydroxyecdysone, 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).

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

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. 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. Accordingly, it is shown that cyst/mmy is required for chitin deposition in the trachea and for the formation of the embryonic cuticle. 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. 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), 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. Shown in this study 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).

Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development

Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, this study demonstrated that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. These findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein (Duan, 2022).


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): 179-193. PubMed ID: 16277981

Duan, Y., Zhu, W., Zhao, X., Merzendorfer, H., Chen, J., Zou, X. and Yang, Q. (2022). Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development. Insect Biochem Mol Biol 141: 103718. PubMed ID: 34982980

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): 423-430. PubMed ID: 16139230

Wang, Y., Norum, M., Oehl, K., Yang, Y., Zuber, R., Yang, J., Farine, J. P., Gehring, N., Flotenmeyer, M., Ferveur, J. F. and Moussian, B. (2020). Dysfunction of Oskyddad causes Harlequin-type ichthyosis-like defects in Drosophila melanogaster. PLoS Genet 16(1): e1008363. PubMed ID: 31929524

Genes involved in tissue development

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