InteractiveFly: GeneBrief

krotzkopf verkehrt: Biological Overview | References

Gene name - krotzkopf verkehrt

Synonyms -

Cytological map position - 83A1-83A1

Function - enzyme

Keywords - a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues - development of ball-and-socket joints in the Drosophila leg - wound repair - extracellular matrix

Symbol - kkv

FlyBase ID: FBgn0001311

Genetic map position - chr3R:5,378,093-5,392,866

Classification - MFS: Major Facilitator Superfamily - Glyco_tranf_GTA_type: Glycosyltransferase family A (GT-A) includes diverse families of glycosyl transferases with a common GT-A type structural fold

Cellular location - Surface transmembrane

NCBI links: EntrezGene, Nucleotide, Protein

Kkv orthologs: Biolitmine
Recent literature
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
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.

The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. This study used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. There is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, cell-cell junctions are key players in this aECM patterning and organisation, and individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, it is proposed that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation (Ozturk-Colak, 2016).

This study examined the apical ECM (aECM) of Drosophila melanogaster trachea, the insect respiratory system. Once the different branches of the tracheal system have been established to cover the overall embryonic body, tracheal cells begin to secrete the components of a chitin-rich aECM that lines up the lumen of the tracheal tubes and can be visualised by the incorporation of chitin-binding probes. A distinctive feature of this aECM are taenidial folds, a series of cuticle ridges that compose a helical structure running perpendicular to the tube length along the entire lumen. Taenidia are believed to confer mechanical strength to the tubes and have been compared to a coiled spring within a rubber tube or to the corrugated hose of a vacuum cleaner. From the very first descriptions, it was noticed that taenidia are unaffected by the presence of cell boundaries, thereby indicating that they are a supracellular structure and suggesting a substantial degree of intercellular coordination. It has been reported that taenidial organisation correlates with that of the apical F-actin bundles in underlying cells—the formation of these bundles preceding the appearance of taenidia. However, the relationship between these bundles and taenidia is still poorly understood. In addition, physical modelling has recently revealed that the interaction of the apical cellular membrane and the aECM determines the stability of biological tubes, thus generating more questions about how this interaction occurs (Ozturk-Colak, 2016).

This study reports that there is a dynamic relationship between sub-apical F-actin and taenidial folds during tracheal lumen formation. Cell-cell junctions participate in organising F-actin bundles and the taenidial fold supracellular aECM and this chitinous aECM contributes to regulating F-actin organisation in a two-way regulatory mechanism (Ozturk-Colak, 2016).

The contribution of chitin deposition to the organisation of taenidia was examined. When studying the contribution of tracheal actin rings to this process, mutants were chosen that do not completely inhibit chitin deposition, as these mutants would probably heavily impair tracheal development, thus hindering specific analysis of the morphogenesis of taenidia. Thus, this investigation turned to Blimp-1, an ecdysone response gene that encodes the Drosophila homolog of the transcriptional factor B-lymphocyte-inducing maturation protein gene and whose mutants have been reported to have misshapen trachea almost completely devoid of taenidia (Ozturk-Colak, 2016).

Indeed, Blimp-1 mutant embryos were grossly inflated compared to the wild-type, a phenotype associated with weaker embryonic cuticles caused by mutations impairing the deposition or organisation of chitin. Consistent with this observation, Blimp-1 mutants showed a pale ectodermal cuticle with smaller denticles, although their phenotype is weaker than that of the kkv chitin synthase mutants. This observation suggests that, while chitin deposition is severely impaired, some still accumulated in the cuticle of Blimp-1 mutant embryos. In support of this hypothesis, lower levels of fluostain signal were detected in the trachea of Blimp-1 mutants compared to the wild-type. Thus, this study expected to find similarly less conspicuous taenidia, which was indeed the case. However, the most obvious abnormal feature of taenidia was their pattern, as they were not organised in folds perpendicular to the tube axis but instead ran parallel to it. Given the close correlation between taenidia and actin bundle organisation, actin arrangement was examined in Blimp-1 mutants, and it was found to be severely impaired. In most Blimp-1 mutants examined, no tracheal actin rings were observed. However, in the mutant embryos in which apical actin bundles were detected, these were oriented in parallel to the tube length like the chitin structures. Thus, as is the case for the other mutant genotypes examined so far, in Blimp-1 embryos the lack of a proper arrangement of taenidial folds correlates with either the absence or abnormal pattern of actin rings (Ozturk-Colak, 2016).

Detailed ultrastructural analysis by TEM confirmed the close interplay between actin and chitin in both tal/pri and Blimp-1 mutants. In wild-type embryos, each taenidium is formed by a plasma membrane protrusion and the taenidia have a regular shape. Arrangement of plasma membrane protrusions in tal/pri and Blimp-1 mutant tracheal cells is irregular. At the end of embryogenesis, whereas the breadth of these taenidia is very constant in wt animals, it is highly variable in tal/pri and Blimp-1 mutants. This result is in line with the finding that proper F-actin ring organisation and chitin deposition are necessary for taenidial morphogenesis (Ozturk-Colak, 2016).

The observation of an effect of a mutation in a gene required for proper chitin arrangement on actin bundling was unexpected. To assess whether the effect of Blimp-1 mutations on actin organisation was indeed a consequence of abnormal chitin deposition in the tracheal cuticle rather than the result of a direct and yet unknown role of Blimp-1 in F-actin bundling, tracheal actin organisation was examined in mutants for kkv, a gene required for chitin morphogenesis only. Surprisingly, kkv mutants also lacked actin rings, thereby indicating a feedback role of proper chitin-mediated tracheal cuticle in F-actin organisation. In addition, F-actin bundles formed normally and thereafter collapsed in kkv mutants. This finding indicates that a proper cuticle is not required for the establishment of the F-actin rings but instead for their maintenance. This implies that proper chitin deposition/organisation contributes to ensure the proper organisation and stability of the apical F-actin rings (Ozturk-Colak, 2016).

How could the apical chitin in the ECM influence actin bundling? It was observed that both kkv and Blimp-1 mutations had an effect on tracheal cell shape. In the wild-type trachea, the cells of the DT were organised such that the longest axis of their apical shape is parallel to the tube axis. However, in both Blimp-1 and kkv mutant trachea, the anteroposterior elongation of the cells of the DT was lost, causing cells to be more square shaped. Thus, it was hypothesised that the change in taenidial orientation in kkv and Blimp-1 mutants could be attributed to the alteration in the overall orientation or shape of the tracheal cells. Interestingly, a modification of cell shape/orientation also occurs in embryos mutant for the Src-family kinase Src42A. However, and as previously reported for F-actin, this study found taenidia to follow the same organisation in Src42A mutant embryos as the wild-type indicating that proper organisation of taenidia can be uncoupled from correct tracheal cell shape/orientation and thus that the former is not merely a consequence of the latter (Ozturk-Colak, 2016).

Having identified and characterised genes that specifically affect taenidial patterning, the individual cell contributions to this supracellular organisation was examined by impairing genetic functions in mosaics. It was not possible to generate mosaics by mitotic recombination since there are no cell divisions after tracheal invagination and RNAi-mediated knockdown often does not work in Drosophila embryogenesis. This was indeed the case upon expression of UAS-RNAi constructs for either tal/pri or Blimp-1 in the embryonic tracheal cells. Thus, alternative approaches were used to produce tracheal cellular chimeras (Ozturk-Colak, 2016).

First, advantage was taken of the effect of Blimp-1 overexpression on taenidial formation. To generate tracheal DTs with distinct cellular composition, an AbdB-Gal4 line was used that drives expression only in the posterior part of the embryo. This approach served as an internal control within the same embryo. Upon expression of UASBlimp-1 under these conditions, lower levels of chitin were detected in the posterior metameres. Thus, chitin deposition seems to be highly dependent on the levels of Blimp-1 activity as both loss-of-function mutations and overexpression of Blimp-1 induce low levels of chitin. It is also noted that overexpression of Blimp-1 gives rise to tracheal cells with a less elongated apical side, like that of Blimp-1 and kkv mutants. Then the trachea at the border of the AbdB-Gal4 domain were examined, finding a perfect correlation between the different physical appearance of taenidia and cells and their genotype, with either wild-type or increased levels of Blimp-1. Flip-out clones expressing Blimp-1 were generated in a wild-type background, and similar results were obtained in these clones. Thus, it is concluded that Blimp-1 regulates chitin accumulation in a cell-autonomous manner and that each cell contributes independently to the chitin deposition of their corresponding segments of the taenidial folds (Ozturk-Colak, 2016).

As a second approach to mosaic analysis, the same AbdBGal4 line was used to drive expression of tal/pri and Blimp-1 in tal/pri and in Blimp-1 loss-of-function mutant backgrounds, respectively. For both mutants, a rescuing effect was seen in the posterior tracheal metameres as taenidial folds became organised perpendicularly to the tube length. Using this approach, it was possible to generate borders of cells with and without tal/pri and Blimp-1 function and taenidia were analyzed in these conditions. In the case of the tal/pri rescue experiment, a difference was detected between the cells expressing the wild-type tal/pri gene and those with a wild-type phenotype, an observation consistent with the non-cell autonomous function of the Tal/Pri peptides. However, in the case of the Blimp-1 rescue experiment, taenidia tended to follow the orientation dictated by the genotype of their respective cells. Moreover, and due to the expression domain of the AbdBGAL4 driver not being completely continuous, single cells of one of the genotypes were observed surrounded by cells of the other and either mutant cells could be detected with a longitudinal arrangement of the taenidia or 'rescued' cells with a perpendicular arrangement; in this case, there was a correlation between the physical appearance of taenidia and the corresponding cell genotype. Interestingly, intermediate orientations between the prototypical longitudinal taenidia were also detected in the mutant domain and the perpendicular ones in the rescued domain. These results suggest that cells 'adapt' the orientation of 'their' segments of the taenidia to the global orientation of the segments of the taenidia contributed by neighbouring cells (Ozturk-Colak, 2016).

These results show that tracheal taenidia can form proper rings even when the neighbouring cells do not. This indicates that, to a certain degree, segments of taenidia can organise properly even in the absence of proper subjacent actin rings provided that the segments of taenidia contributed by the neighbouring cells are properly organised (Ozturk-Colak, 2016).

The role for the apical chitin ECM in tracheal actin organisation indicates a feedback mechanism to generate the supracellular taenidial structures. In the light of the above and previously published results, the following model is proposed for the formation of the taenidial folds that expand the overall diameter of the tracheal tube. On the one hand, actin polymerises in rings at the apical side of the tracheal cells in a tal/pri-dependent process; these actin rings are then required for the particular accumulation of the kkv chitin synthase and for the appearance of folds in the plasma membrane. In turn, kkv accumulation leads to a localised increased production and deposition of chitin along specific enriched stripes above the actin rings in a Blimp-1-mediated process. On the other hand, the cellular AJs are instrumental in ensuring that apical F-actin bundles from each cell follow a supracellular organ arrangement. It has to be noted that each cell appears to independently organise or maintain, to a certain degree, the proper orientation of their actin bundles, as determined by Blimp-1 clonal analysis and the disruption of cell adhesion by downregulation of α-Cat and, consequently, DE-Cad. These results further suggest cell polarity along the circumferential axis of the tracheal tube. Nevertheless, this is not an absolute value as cells also have the capacity to modify the orientation of their sections of the taenidia to keep the continuity of these structures along the tube. In this regard, cell adhesion is central to ensure the continuity of the intracellular actin bundles as a patterning element for the overall tube. Subsequently, the chitin aECM feeds back on to the cellular architecture by stabilising F-actin bundling and cell shape via the modulation of Src42A phosphorylation levels. The combination of all these phenomena explain just how it is that the cells of the tracheal epithelium can cooperate unconsciously so as to form a helicoid [chitinous] thickening continuous from one end of the trachea to another (Ozturk-Colak, 2016).

Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila

Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. This study reports the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and six genes were identified as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101 (Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas disoriented taenidial folds were observed on larval tracheae when Lines (lin) or Autophagy-related 3 (Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr and Abd-A showed the potential complex mechanism of kkv transcription are regulated by an interaction network with bithorax complex components. This study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis (Yue, 2021).

Chitinase10 controls chitin amounts and organization in the wing cuticle of Drosophila

Wings are essential for insect fitness. A number of proteins and enzymes have been identified to be involved in wing terminal differentiation, which is characterized by the formation of the wing cuticle. This study addressed the question whether Chitinase 10 (Cht10) may play an important role in chitin organization in the wings of the fruit fly Drosophila melanogaster. Cht10 expression was found to coincide with the expression of the chitin synthase coding gene kkv. This suggests that the respective proteins may cooperate during wing differentiation. In tissue-specific RNA interference experiments, it was demonstrated that suppression of Cht10 causes an excess in chitin amounts in the wing cuticle. Chitin organization is severely disrupted in these wings. Based on these data, it is hypothesized that Cht10 restricts chitin amounts produced by Kkv in order to ensure normal chitin organization and wing cuticle formation. In addition, it was found by scanning electron microscopy that Cht10 suppression also affects the cuticle surface. In turn, cuticle inward permeability is enhanced in Cht10-less wings. Moreover, flies with reduced Cht10 function are unable to fly. In conclusion, Cht10 is essential for wing terminal differentiation and function (Dong, 2020).

Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis

Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. This study uncovered a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, both substitutions (I1056M/F) were introduced in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro (Douris, 2016).

Deciphering the genetic programme triggering timely and spatially-regulated chitin deposition

Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. This study addressed this issue by analysing the deposition of the chitinous extracellular matrix of Drosophila, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). This process was shown to equally require the activity of two other genes, namely expansion (exp) and rebuf (reb; CG13183). Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, these results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. This mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, it was observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, this study has identified the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity (Moussian, 2015).

Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg

Animal body shape is framed by the skeleton, which is composed of extracellular matrix (ECM). Although how the body plan manifests in skeletal morphology has been studied intensively, cellular mechanisms that directly control skeletal ECM morphology remain elusive. In particular, how dynamic behaviors of ECM-secreting cells, such as shape changes and movements, contribute to ECM morphogenesis is unclear. Strict control of ECM morphology is crucial in the joints, where opposing sides of the skeleton must have precisely reciprocal shapes to fit each other. This study found that, in the development of ball-and-socket joints in the Drosophila leg, the two sides of chitin-based ECM form sequentially. Distinct cell populations produce the 'ball' and the 'socket', and these cells undergo extensive shape changes while depositing ECM. It is proposed that shape changes of ECM-producing cells enable the sequential ECM formation to allow the morphological coupling of adjacent components. These results highlight the importance of dynamic cell behaviors in precise shaping of skeletal ECM architecture (Tajiri, 2010).

This study revealed that the ball and the socket cuticles develop sequentially. The ball-producing activity and the socket-producing activity are allocated to distinct cell populations, and have found that shape changes of these cells that occur simultaneously with their cuticle-secreting activities result in the interlocking ball-and-socket structure. As the ball cuticle builds up, concurrent cell shape changes drive the apical domains of ball-producing cells out of the cavity and bring in the apical domains of the socket-producing cells, resulting in close enwrapment of the ball by the latter cell population. Accordingly, the shape of the resulting socket cuticle conforms to that of the ball. Synchronization between ECM formation and dynamic relocation of the cell surfaces that mediate it thus underlies the building of the complex ECM structure (Tajiri, 2010).

A map of ball-producing and socket-producing cells best summarizes the results of krotzkopf verkehrt (kkv - encoding Chitin Synthase 1) RNAi, and is consistent with the result indicating their continuous association with respective parts of the cuticle during their formation. The ball morphology was severely disrupted by bib>kkv RNAi but not by neur>kkv RNAi, indicating that the ball-producing activity is restricted to the distal subset of bib-expressing cells that do not significantly express neur. Consistently, these cells are in constant contact with the ball cuticle throughout its formation. The cuticle phenotype of neur>kkv RNAi shows that neur-expressing cells are responsible for forming the ventral part of the socket cuticle, with which they continue to associate. Likewise, fng-expressing cells contribute to the formation of the remainder of the socket. Partial disruption of the socket by bib>kkv RNAi should be, to some extent, due to direct blocking of socket production in cells co-expressing bib and neur. Additionally, the impairment of ball formation might somehow interfere with socket formation. Occasional deformation of the ball by neur>kkv RNAi might be caused by marginal expression of neur in the presumptive ball-producing cells (Tajiri, 2010).

Patterns of ECM-producing tissues do play a major role in the regulation of ECM morphology. Previous studies have unraveled how global positional information affects skeletal patterns through the regulation of specification, differentiation and proliferation of ECM-producing cells. There, the morphology of ECM was assumed to be synonymous with that of the cells or tissues that secrete it. The present study illustrates that the skeletal morphology reflects not only the pattern of those cells at one point in time, but also the history of their dynamic behaviors during ECM formation. Secreted apically by the epidermis, the cuticle is monolayered in most parts. In the joints, however, relocation of the secretory surfaces enables formation of a cuticle beneath a pre-formed layer. Cell motility thus allows a tissue of simple configuration to build a complex and essential three-dimensional ECM structure. It is envisioned that movements of ECM-secreting cells probably play important roles in ECM morphogenesis in other systems, especially in formation or adjustment of intricate skeletal structures (Tajiri, 2010).

The morphology of the cuticle, as well as how it develops, correlated well with cell shape changes. This suggested either that the cell-shape changes govern the morphology of the cuticle, and/or vice versa. This study found that the movement of the apical surfaces of the cells was correctly oriented even when the shape of the cuticle was disrupted, indicating that the morphogenesis of the ball-and-socket cuticle is primarily controlled by the way the cells change their shapes as they deposit the cuticle. How do the cells know which way to move? In other words, what is the molecular mechanism that mediates global proximodistal polarity of the leg to direct cell movement? In mutants of well-known planar cell polarity genes, such as frizzled, dishevelled and prickled, extra joints of reverse proximodistal polarity are formed. Nonetheless, the ball-and-socket structure of individual joints remains largely intact, indicating that cell shape changes are correctly guided by a mechanism other than this pathway. Analysis and local disruption of cytoskeletal architecture in the joint region could help answer this question (Tajiri, 2010).

These results do not rule out the possibility that the cuticle plays a permissive role in cell movements. The ECM generally affects cell shape and motility, and chitin-based ECM has been shown to regulate epithelial morphogenesis in some Drosophila tissues. Whether the cuticle provides a permissive environment for cell shape changes in the joint is an important issue to address in future work (Tajiri, 2010).

The formation of reciprocally shaped interfaces is vital for the sake of joint function. The serial progression of ball-and-socket morphogenesis shown in this study can be compared to ‘mold casting’: (1) the ball enlarges rapidly through stratification, and the cavity expands to accommodate it, and (2) the enlarged cavity then serves as the 'mold' along which the socket cuticle is formed. Hence, the shape of the ball is transmitted to the socket (the 'cast'). Whether or not this model also applies to vertebrate synovial joints is an intriguing question. It has been speculated that, in the chick digit joints, chondrogenic cell differentiation on the distal side might promote its expansion to become convex; at the same time, proliferation of peripheral cells on the proximal side might permit them to grow and wrap themselves around the distal side, thereby becoming concave. If this were the case, that model can be regarded as a modified version of ball-and-socket morphogenesis, one side fitting to the other through cell proliferation instead of cell shape changes. It will then become important to study how cells and ECM collectively undergo morphogenesis in other types of joints and in other species. Unraveling similarities and differences in the modes of joint development would be crucial in a medical sense as well, for understanding various joint pathologies and designing therapies to treat them (Tajiri, 2010).

Multiple transcription factor codes activate epidermal wound-response genes in Drosophila

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. This study characterized the transcriptional regulatory enhancers of 4 genes-Ddc, ple, msn, and kkv-that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. It is proposed that the combination of GRH and FOS is part of an ancient wound-response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. The results indicate that this is an overly simplistic view (Pearson, 2009).

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 (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, 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 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 (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 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).

Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster

Exoskeletons stabilize cell, tissue, and body morphology in many living organisms including fungi, plants, and arthropods. In insects, the exoskeleton, the cuticle, is produced by epidermal cells as a protein extracellular matrix containing lipids and the polysaccharide chitin, and its formation requires coordinated synthesis, distribution, and modification of these components. Eventually, the stepwise secretion and sorting of the cuticle material results in a layered structure comprising the envelope, the proteinaceous epicuticle, and the chitinous procuticle. To study the role of chitin during cuticle development, this study analyzed the consequences of chitin absence in the embryo of Drosophila melanogaster caused by mutations in the Chitin Synthase-1 (CS-1) gene, called krotzkopf verkehrt (kkv). Histological data confirm that chitin is essential for procuticle integrity and further demonstrate that an intact procuticle is important to assemble and to stabilize the chitin-less epicuticle. Moreover, the phenotype of CS-1/kkv mutant embryos indicates that chitin is required to attach the cuticle to the epidermal cells, thereby maintaining epidermal morphology. Finally, sclerotization and pigmentation, which are the last steps in cuticle differentiation, are impaired in tissues lacking CS-1/kkv function, suggesting that proper cuticle structure is crucial for the activity of the underlying enzymes (Moussian, 2005).

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster

The embryonic cuticle of Drosophila is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. Mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle have been characterized. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion being reversed within the eggshell (the 'retroactive' phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the 'blimp' phenotype). One of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. Two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and a new mutation, called zeppelin (zep), is described that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do (Ostrowski, 2002).

The cuticle defects, particularly the disruption of the head skeleton, are most severe in kkv and grh mutants. All alleles of kkv, both those isolated previously and those identified in this screen, produce similar phenotypes. When removed from the vitelline membrane, kkv and grh mutant embryos are very flaccid and are not motile although they are able to contract their body wall muscles. All three alleles of knk and the one available allele of rtv produce milder defects in the head skeleton and denticle belts. When removed from the vitelline membrane they are more robust than the kkv and grh mutants, and they are motile but die within hours after removal from the eggshell. The head skeleton and denticle belts of zep mutants are almost wild type and these embryos are sometimes able to hatch on their own, although they die at roughly the same stage as the knk and rtv mutants. The degree of cuticle expansion can vary among cuticle preparations due to uncontrollable differences in the mechanical devitellinization process. However, the severity of head defects, flaccidity, and motility are consistent within the alleles of each complementation group. Thus the phenotypic effects of the blimp class mutations can be ranked from most to least severe: kkv, grh > rtv, knk > zep (Ostrowski, 2002).

The identification of kkv as a chitin synthase and the ability of a chitin synthesis inhibitor to phenocopy kkv shows that disrupting synthesis or deposition of chitin alone can account for the blimp phenotype. However, it is believed that two of the blimp class genes, knk and zep, may function in the epidermis prior to cuticle deposition because both interact genetically with mutations in the Drosophila E-cadherin, encoded by shotgun (Ostrowski, 2002).

grainy head affects head skeleton and embryonic cuticle. grh encodes a GATA family transcription factor and activates the transcription of a number of genes during development, one of which is Dopa-decarboxylase (Ddc). This enzyme is synthesized in the cuticle-secreting layer of cells at the end of embryogenesis; the dopamine produced undergoes further metabolism and oxidation to produce quinones that crosslink cuticular proteins. Thus, loss of grh function would result in weakening of the cuticle indirectly through its failure to activate Ddc expression (Ostrowski, 2002).

Stage-specific expression of the chitin synthase DmeChSA and DmeChSB genes during the onset of Drosophila metamorphosis

Chitin, the major structural polysaccharide of arthropods, is an important constituent of the insect extracellular structures, cuticle and gut peritrophic matrix. Synthesis of cuticular chitin is strictly coordinated with the ecdysone-regulated molting cycle of insect development (the term "ecdysone" is used in this paper instead of "ecdysteroids" since the exact ratio of various hormonal forms changes during metamorphosis). Based on observed similarities between the fungal chitin synthases and other processive beta-glycosyltransferases, This study has identified the first insect chitin synthase genes, DmeChSA and DmeChSB/Kkv (Database accession numbers: EMBL/GenBank/DDBJ A83122, A83126, AJ309488, AJ309489), from Drosophila melanogaster. Chromosomal localization has identified these genes close to and on either side of the centromere of the third chromosome. Partial cDNA clones of both genes have been isolated from a pupal cDNA library. To obtain the first insight into the transcriptional regulation of chitin synthesis, the expression of DmeChSA and DmeChSB was monitored during the periods of the late-larval and prepupal ecdysone pulses that direct metamorphosis. Transcripts of either gene are barely detected prior to and during the late-larval ecdysone pulse. Once the late-larval ecdysone pulse is ceased completely, both DmeChSA and DmeChSB genes show a remarkable up-regulation (Gagou, 2002).


Search PubMed for articles about Drosophila Kkv

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

Dong, W., Gao, Y. H., Zhang, X. B., Moussian, B. and Zhang, J. Z. (2020). Chitinase10 controls chitin amounts and organization in the wing cuticle of Drosophila. Insect Sci. PubMed ID: 32129536

Douris, V., Steinbach, D., Panteleri, R., Livadaras, I., Pickett, J. A., Van Leeuwen, T., Nauen, R. and Vontas, J. (2016). Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis. Proc Natl Acad Sci U S A 113(51): 14692-14697. PubMed ID: 27930336

Gagou, M. E., Kapsetaki, M., Turberg, A. and Kafetzopoulos, D. (2002). Stage-specific expression of the chitin synthase DmeChSA and DmeChSB genes during the onset of Drosophila metamorphosis. Insect Biochem Mol Biol 32(2): 141-146. PubMed ID: 11755055

Moussian, B., Letizia, A., Martinez-Corrales, G., Rotstein, B., Casali, A. and Llimargas, M. (2015). Deciphering the genetic programme triggering timely and spatially-regulated chitin deposition. PLoS Genet 11: e1004939. PubMed ID: 25617778

Moussian, B., Schwarz, H., Bartoszewski, S. and Nusslein-Volhard, C. (2005). Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster. J Morphol 264(1): 117-130. PubMed ID: 15747378

Ostrowski, S., Dierick, H. A. and Bejsovec, A. (2002). Genetic control of cuticle formation during embryonic development of Drosophila melanogaster. Genetics 161: 171-182. PubMed ID: 12019232

Ozturk-Colak, A., Moussian, B., Araujo, S. J. and Casanova, J. (2016). A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea. Elife 5. PubMed ID: 26836303

Pearson, J. C., Juarez, M. T., Kim, M., Drivenes, O. and McGinnis, W. (2009). Multiple transcription factor codes activate epidermal wound-response genes in Drosophila. Proc Natl Acad Sci U S A 106(7): 2224-2229. PubMed ID: 19168633

Tajiri, R., Misaki, K., Yonemura, S. and Hayashi, S. (2010). Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg. Development 137(12): 2055-63. PubMed ID: 20501594

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

Yue, X., Liang, Y., Wei, Z., Lv, J., Cai, Y., Fan, X., Zhang, W. and Chen, J. (2021). Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila. Insect Sci. PubMed ID: 34351065

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date revised: 4 April 2022

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