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Tracheal branching morphogenesis in Drosophila: New insights into cell behaviour and organ architecture
Abstract
Our understanding of the molecular control of morphological processes has increased tremendously over recent years through the development and use of high resolution in vivo imaging approaches, which have enabled cell behaviour to be linked to molecular functions. Here we review how such approaches have furthered our understanding of tracheal branching morphogenesis in Drosophila, during which the control of cell invagination, migration, competition and rearrangement is accompanied by the sequential secretion and resorption of proteins into the apical luminal space, a vital step in the elaboration of the trachea's complex tubular network. We also discuss the similarities and differences between flies and vertebrates in branched organ formation that are becoming apparent from these studies.
... This convergence-extension process is orchestrated by contractile actomyosin cables that induce directed cell rearrangements, similar to what was observed during vertebrate neurulation [8]. Other morphogenetic events, such as the formation of tubular structures of trachea and salivary glands, involve both apical cell constrictions and directed cell rearrangements, relying on combinations of these dynamic building blocks [8][9][10][11]. Thus, morphogenetic processes appear to be assembled from commonly used morphogenetic motifs, including apical cell constrictions, cell intercalations, delaminations, collective divisions, and migrations [12][13][14][15][16][17]. ...
... Then, similarly to a mathematical induction, by inferring the spatial correspondence between slices at consecutive time points cell indices can be propagated from one time point to the next. To perform the spatial matching we used Maxwell's demons algorithm for non-rigid registration [59] (matlab implementation of "imregdemons" with three pyramid levels, for which the number of iterations was: [10,20,30]). Since the binarized slices are relatively poor in spatial information, the matching was calculated on the raw microscopy slices and applied on the binarized slices. ...
Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. Here, we present a “template-based search” approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. We formulated the task of motif mapping as a subsequence matching problem and solved it using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows our algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate our approach, we mapped cell intercalations during germband extension in the early Drosophila embryo. Our framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. Our formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.
... AWD was the first NDPK whose role was examined in in vivo cell migration models: AWD's function was investigated in two well characterised migrating cell types during Drosophila development: in migration of tracheal cells and border cells. Fly tracheogenesis is a model of tubular morphogenesis [80,81]. The process starts during early embryogenesis by emergence of tracheal placodes, an invagination of specialized ectodermal cells, from which tracheal branches later develop. ...
... Cell migration and fusion events will give rise to the tracheal network as tracheal branches elongate, migrate and fuse to form tracheal tubules (branching morphogenesis). FGF (fibroblast growth factor) is the key diffusive chemotactic signal, which drives tracheal cell migration: migrating tracheal cells express fibroblast growth factor receptor (FGFR) on their surface and follow FGF, the signal released by the surrounding tissues [80]. AWD was found to negatively regulate tracheal migration with low AWD dosage correlating with ectopic migration of tracheal cells. ...
Metastasis suppressor genes (MSGs) inhibit different biological processes during metastatic progression without globally influencing development of the primary tumor. The first MSG, NM23 (non-metastatic clone 23, isoform H1) or now called NME1 (stands for non-metastatic) was identified some decades ago. Since then, ten human NM23 paralogs forming two groups have been discovered. Group I NM23 genes encode enzymes with evolutionarily highly conserved nucleoside diphosphate kinase (NDPK) activity. In this review we summarize how results from NDPKs in model organisms converged on human NM23 studies. Next, we examine the role of NM23-H1 and its homologs within the metastatic cascade, e.g. cell migration and invasion, proliferation and apoptosis. NM23-H1 homologs are well known inhibitors of cell migration. Drosophila studies revealed that AWD, the fly counterpart of NM23-H1 is a negative regulator of cell motility by modulating endocytosis of chemotactic receptors on the surface of migrating cells in cooperation with Shibire/Dynamin; this mechanism has been recently confirmed by human studies. NM23-H1 inhibits proliferation of tumor cells by phosphorylating the MAPK scaffold, kinase suppressor of Ras (KSR), resulting in suppression of MAPK signalling. This mechanism was also observed with the C. elegans homolog, NDK-1, albeit with an inverse effect on MAPK activation. Both NM23-H1 and NDK-1 promote apoptotic cell death. In addition, NDK-1, NM23-H1 and their mouse counterpart NM23-M1 were shown to promote phagocytosis in an evolutionarily conserved manner. In summary, inhibition of cell migration and proliferation, alongside actions in apoptosis and phagocytosis are all mechanisms through which NM23-H1 acts against metastatic progression.
... Organ system development across higher order species requires formation of tubular networks. These networks can be found in the respiratory system (Affolter and Caussinus, 2008), in the vertebrate ureteric system (Costantini, 2006), and most prominently, in the circulatory system, including the blood and lymphatic vasculature (Horowitz and Simons, 2008). The architecture, and therefore function of such systems, is largely determined by one key topographical feature: branching, which occurs by the sprouting of new tubes from preexisting ones. ...
Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.
... Much of what we know regarding the role of directional migration in branching morphogenesis, including epithelial branching, has been based on studies from invertebrate systems, particularly the fly trachea and air sacs (Lu et al., 2006;Affolter et al., 2003;Affolter and Caussinus, 2008;Ghabrial et al., 2003;Metzger and Krasnow, 1999). In both of these systems, branching does not occur if directional migration of the tracheal or air sac epithelium fails to take place ( Figure 6C) (Sato and Kornberg, 2002;Cabernard and Affolter, 2005;Cabernard et al., 2004). ...
Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin β1 and inhibits its downstream signaling, and Integrin β1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin β1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.
... The Drosophila tracheal system is a highly ramified epithelial tubular network that transports oxygen to internal organs. Its branching pattern is highly stereotyped 194 and is driven by sources of FGF 195 as a guidance cue. Tracheal development begins with the invagination of 20 epithelial sacs, each sprouting finer branches to generate a tree-like structure. ...
... Branching morphogenesis has been studied in model organisms other than the mouse, rat and chick (Affolter and Caussinus, 2008;Bracken et al., 2008;Horowitz and Simons, 2008;Shifley et al., 2012;Rankin et al., 2015). While orthologs of many genes involved in branching have similar roles in other species, these are not within the scope of this review. ...
A common developmental process, called branching morphogenesis, generates the epithelial trees in a variety of organs, including the lungs, kidneys, and glands. How branching morphogenesis can create epithelial architectures of very different shapes and functions remains elusive. In this review, we compare branching morphogenesis and its regulation in lungs and kidneys and discuss the role of signaling pathways, the mesenchyme, the extracellular matrix, and the cytoskeleton as potential organ-specific determinants of branch position, orientation, and shape. Identifying the determinants of branch and organ shape and their adaptation in different organs may reveal how a highly conserved developmental process can be adapted to different structural and functional frameworks and should provide important insights into epithelial morphogenesis and developmental disorders.
... During the formation of the dorsal branch (DB), individual DBs initially elongate as a cluster of cells, led by migrating distal-most cells at the front (tip cells), which induces tension on stalk cells trailing in the back, which ultimately results in stalk cell intercalation (SCI) and elongation of stalk cells 82,86 . SCI occurs in all primary branches except for the ones that will eventually form the dorsal trunk, the largest tube in the tracheal system [86][87][88] . Surprisingly, and in contrast to many other morphogenetic events of tissue elongation through cell intercalation [34][35][36][37][38][39][40][41][42][43] , DB cell intercalation occurs in the absence of MyoII function; intercalation is thus a passive event with regard to force generation in stalk cells, and it is possible that stalk cells need to downregulate actomyosin activity to give in to the tension created by the migration of tip cells 89 . ...
Reversible protein phosphorylation by kinases in extensively used to control a plethora of processes essential for proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases for direct, robust and tissue-specific phosphorylation of target fluorescent protein fusions in vivo. We show that synthetic Rok kinases, based on the Drosophila ortholog of Rho-associated protein kinase (ROCK), are functional enzymes and can activate myosin II through phosphorylation of Sqh::GFP or Sqh::mCherry in different morphogenetic processes in a developing fly embryo. We next use the system to study the impact of actomyosin activation specifically in the developing tracheal branches and showed that ectopic activation of actomyosin with engineered Rok kinase did not prevent cell intercalation nor the formation of autocellular junctions. We assume that this approach can be adapted to other kinases and targets in various eukaryotic genetic systems.
... Epithelial branching in lungs and kidneys is accompanied by overall organ growth, while the stroma in the mammary gland is already established and does not substantially change in size when the epithelium starts to invade it (Sternlicht et al. 2006). Branching morphogenesis has been studied in model organisms other than the mouse, rat and the chick (Affolter and Caussinus 2008;Horowitz and Simons 2008;Shifley et al. 2012;Rankin et al. 2015;Bracken et al. 2008). While orthologs of many genes involved in branching have similar roles in other species, these are not within the scope of this review. ...
A common developmental process, called branching morphogenesis, generates the epithelial trees in a variety of organs, including the lungs, kidneys, and glands. How branching morphogenesis can create epithelial architectures of very different shapes and functions remains elusive. In this review, we compare branching morphogenesis and its regulation in lungs and kidneys and discuss the role of signaling pathways, the mesenchyme, the extracellular matrix, and the cytoskeleton as potential organ-specific determinants of branch position, orientation, and shape. Identifying the determinants of branch and organ shape and their adaptation in different organs may reveal how a highly conserved developmental process can be adapted to different structural and functional frameworks and should provide important insights into epithelial morphogenesis and developmental disorders.
... Individual domain arrangement, which includes signal-peptides, catalytic-, and transmembrane-domains and chitin-binding domains, may cause functional differences among insect glycosylhydrolases family 18. Thus, members can be classified into eight groups 34 : The Drosophila Chitinases contain single (Cht2,4,5,6,8,9,11,12) or more (Cht7, Cht1/Cht3/Cht10) catalytic Glyco18 domains, some possess a chitin-binding domain (Cht5,7,12) others a transmembrane domain (Cht7). In contrast, as Idgf proteins (group V) contain one Glyco18 domain, which lacks a critical glutamate residue, they do not possess chitinolytic activity 40 . ...
Tight barriers are crucial for animals. Insect respiratory cells establish barriers through their extracellular matrices. These chitinous-matrices must be soft and flexible to provide ventilation, but also tight enough to allow oxygen flow and protection against dehydration, infections, and environmental stresses. However, genes that control soft, flexible chitin-matrices are poorly known. We investigated the genes of the chitinolytic glycosylhydrolase-family 18 in the tracheal system of Drosophila melanogaster. Our findings show that five chitinases and three chitinase-like genes organize the tracheal chitin-cuticles. Most of the chitinases degrade chitin from airway lumina to enable oxygen delivery. They further improve chitin-cuticles to enhance tube stability and integrity against stresses. Unexpectedly, some chitinases also support chitin assembly to expand the tube lumen properly. Moreover, Chitinase2 plays a decisive role in the chitin-cuticle formation that establishes taenidial folds to support tube stability. Chitinase2 is apically enriched on the surface of tracheal cells, where it controls the chitin-matrix architecture independently of other known cuticular proteins or chitinases. We suppose that the principle mechanisms of chitin-cuticle assembly and degradation require a set of critical glycosylhydrolases for flexible and not-flexible cuticles. The same glycosylhydrolases support thick laminar cuticle formation and are evolutionarily conserved among arthropods.
... Misregulation of tube development or maintenance can have severe implications, including polycystic kidney disease in humans (Harris and Torres, 2009;Song et al., 2017). However, the mechanisms by which small tubes control their lengths and diameters is largely unknown (reviewed by Affolter and Caussinus, 2008;Iruela-Arispe and Beitel, 2013;Wang et al., 2017). Even basic descriptions of how cell shape and organization change during development, which is a fundamental prerequisite for understanding size control mechanisms, has not been determined for most tubes. ...
Biological tubes are essential for animal survival, and their functions are dependent on tube shape. Analyzing the contributions of cell shape and organization to the morphogenesis of small tubes has been hampered by the limitations of existing programs in quantifying cell geometry on highly curved tubular surfaces and calculating tube-specific parameters. We therefore developed QuBiT (Quantitative Tool for Biological Tubes) and used it to analyze morphogenesis of the embryonic Drosophila trachea (airway). In the main tube, we find previously unknown anterior-to-posterior (A-P) gradients of cell apical orientation and aspect ratio, and periodicity in the organization of apical cell surfaces. Inferred cell intercalation during development dampens an A-P gradient of the number of cells per cross-section of the tube, but does not change the patterns of cell connectivity. Computationally 'unrolling' the apical surface of wild-type trachea and the hindgut reveals previously unrecognized spatial patterns of the apical marker Uninflatable and a non-redundant role for the Na+/K+ ATPase in apical marker organization. These unexpected findings demonstrate the importance of a computational tool for analyzing small diameter biological tubes.
... Collective cell migration is a major mode of cell migration that leads to tissue rearrangement during developmental morphogenesis. In fact, its underlying mechanism and function have been well studied using various animal models, such as tracheal network branching and border cell migration in Drosophila embryos (1,31), migration of primordium of the mechanosensory lateral line organ in zebrafish embryos (6) and neural crest cell migration in zebrafish, Xenopus, chick and mouse embryos (32). Collective cell migration has also been implicated in wound healing of both embryos (33) and adult tissues (34). ...
Much attention has been paid on the mechanism of cancer invasion from the viewpoint of the behavior of individual cancer cells. On the other hand, histo-pathological analyses of specimens from cancer patients and of cancer invasion model animals have revealed that cancer cells often exhibit collective invasion, characterized by sustained cell-to-cell adhesion and polarized invasion as cell clusters. Interestingly, it has recently become evident that during collective invasion of cancer cells the cells localized at invasion front (leader cells) and the cells following them (follower cells) exhibit distinct cellular characteristics, and that there exist the cells expressing representative proteins related to both epithelial and mesenchymal properties simultaneously, designated as hybrid epithelial-to-mesenchymal transition (EMT)-induced cells, in cancer tissue. Furthermore, the findings that cells adopted in hybrid EMT state form clusters and show collective invasion in vitro emphasize an importance of hybrid EMT-induced cells in collective cancer invasion. In this article, we overview recent findings of the mechanism underlying collective invasion of cancer cells and discuss the possibility of controlling cancer invasion and metastasis by targeting this process.
The unique design of respiratory organs in multicellular organisms makes them prone to infection by pathogens. To cope with this vulnerability, highly effective local immune systems evolved that are also operative in the tracheal system of insects. Many pathogens and parasites (including viruses, bacteria, fungi, and metazoan parasites) colonize the trachea or invade the host via this route. Currently, only two modules of the tracheal immune system have been characterized in depth: 1) Immune deficiency pathway-mediated activation of antimicrobial peptide gene expression and 2) local melanization processes that protect the structure from wounding. There is an urgent need to increase our understanding of the architecture of tracheal immune systems, especially regarding those mechanisms that enable the maintenance of immune homeostasis. This need for new studies is particularly exigent for species other than Drosophila.
Drosophila trachea is a classical model for analyzing epithelial, especially tubular epithelial biology. We identify lateral E-cadherin mediated junctions that encircle the cells just basal to the zonula adherens in the larval trachea. The lateral junction is associated with downstream adapters, including catenins, and has a distinct junctional actin cortex. The lateral cortex contributes to the development of a supracellular actomyosin mesh in the late larvae. Establishment of this cytoskeletal structure depends on lateral junction associated Rho1 and Cdc42 GTPases, and Arp and WASP pathways. The supracellular network takes the character of stress fibers along the AP axis in the early hours of pupation. It contributes to the shortening of the epithelial tube albeit in a manner redundant to ECM-mediated compression mechanism. In conclusion, we show the in vivo existence of functional lateral adherens junction and suggest a role for it in mediating dynamic cytoskeletal events during tissue scale morphogenesis.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
The Insects has been the standard textbook in the field since the first edition published over forty years ago. Building on the strengths of Chapman's original text, this long-awaited 5th edition has been revised and expanded by a team of eminent insect physiologists, bringing it fully up-to-date for the molecular era. The chapters retain the successful structure of the earlier editions, focusing on particular functional systems rather than taxonomic groups and making it easy for students to delve into topics without extensive knowledge of taxonomy. The focus is on form and function, bringing together basic anatomy and physiology and examining how these relate to behaviour. This, combined with nearly 600 clear illustrations, provides a comprehensive understanding of how insects work. Now also featuring a richly illustrated prologue by George McGavin, this is an essential text for students, researchers and applied entomologists alike.
Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase–substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase–substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.
Biological tubes are fundamental units of most metazoan organs. Their defective morphogenesis can cause malformations and pathologies. An integral component of biological tubes is the extracellular matrix, present apically (aECM) and basally (BM). Studies using the Drosophila tracheal system established an essential function for the aECM in tubulogenesis. Here, we demonstrate that the BM also plays a critical role in this process. We find that BM components are deposited in a spatial-temporal manner in the trachea. We show that laminins, core BM components, control size and shape of tracheal tubes and their topology within the embryo. At a cellular level, laminins control cell shape changes and distribution of the cortical cytoskeleton component α-spectrin. Finally, we report that the BM and aECM act independently—yet cooperatively—to control tube elongation and together to guarantee tissue integrity. Our results unravel key roles for the BM in shaping, positioning, and maintaining biological tubes.
Branching networks are a very common feature of multicellular animals and underlie the formation and function of numerous organs including the nervous system, the respiratory system, the vasculature and many internal glands. These networks range from subcellular structures such as dendritic trees to large multicellular tissues such as the lungs. The production of branched structures by single cells, so called subcellular branching, which has been better described in neurons and in cells of the respiratory and vascular systems, involves complex cytoskeletal remodelling events. In Drosophila, tracheal system terminal cells (TCs) and nervous system dendritic arborisation (da) neurons are good model systems for these subcellular branching processes. During development, the generation of subcellular branches by single-cells is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. In this review, we describe the current knowledge on cytoskeletal regulation of subcellular branching, based on the terminal cells of the Drosophila tracheal system, but drawing parallels with dendritic branching and vertebrate vascular subcellular branching.
In many developmental contexts, epithelial folding is accompanied by neighboring tissue flows towards the invagination site. To assess the idea of a long-range coupling between invagination and tissue flow, we have studied two macroscopic morphogenetic events happening during Drosophila metamorphosis: the invagination of the head-to-thorax boundary and the anterior dorsal thorax flow. Using large-scale laser ablations or locally disrupting MyosinII contractility in the invaginating region, we have found that head-thorax interface invagination is an active process which is providing a pulling force locally driving thorax flow. However, we have discovered that thorax flow is also partly autonomous, and thereby can actively provide a pushing force towards the invagination site. Using forward genetic methods, we have uncovered that thorax flow rely on collective migration on the apical extracellular matrix (ECM), depending on the secretion of the ZP protein Dumpy and on planar polarity cues. We have furthermore shown in vivo that regions of active thorax migration are specifically associated with low apical ECM deformation. Taken together, my PhD work uncovers a new mechanism coordinating tissue folding and tissue flow, and emphasizes the importance of tissue-matrix interaction in epithelial mechanics and morphogenesis.
Tissue rearrangements that are essential for several physiological and pathological conditions depend on the coordinated migration of cellular clusters. The success of these clusters in reaching their target tissues relies on their ability to migrate in a synchronized manner. An outstanding feature of collectively migrating cells is their high degree of migratory efficiency. A large body of evidence shows that this property emerges from fine-tuning the mechanical coupling between neighbor cells, which in turn synchronizes both fluent molecular communication and efficient synchronization of cytoskeletal activities of collectively migrating cells. This exquisite communication ensures efficient transmission of signals from the microenvironment and across the cluster. This highly regulated, cooperative, and peculiar mode of cellular motion is defined as collective cell migration (CCM) and due to its relevance for embryogenesis, wound healing, regeneration, and cancer metastasis, CCM is currently a research hotspot.
How the shape of embryos and organs emerges during development is a fundamental question that has fascinated scientists for centuries. Tissue dynamics arise from a small set of cell behaviours, including shape changes, cell contact remodelling, cell migration, cell division and cell extrusion. These behaviours require control over cell mechanics, namely active stresses associated with protrusive, contractile and adhesive forces, and hydrostatic pressure, as well as material properties of cells that dictate how cells respond to active stresses. In this Review, we address how cell mechanics and the associated cell behaviours are robustly organized in space and time during tissue morphogenesis. We first outline how not only gene expression and the resulting biochemical cues, but also mechanics and geometry act as sources of morphogenetic information to ultimately define the time and length scales of the cell behaviours driving morphogenesis. Next, we present two idealized modes of how this information flows — how it is read out and translated into a biological effect — during morphogenesis. The first, akin to a programme, follows deterministic rules and is hierarchical. The second follows the principles of self-organization, which rests on statistical rules characterizing the system’s composition and configuration, local interactions and feedback. We discuss the contribution of these two modes to the mechanisms of four very general classes of tissue deformation, namely tissue folding and invagination, tissue flow and extension, tissue hollowing and, finally, tissue branching. Overall, we suggest a conceptual framework for understanding morphogenetic information that encapsulates genetics and biochemistry as well as mechanics and geometry as information modules, and the interplay of deterministic and self-organized mechanisms of their deployment, thereby diverging considerably from the traditional notion that shape is fully encoded and determined by genes.
The hedgehog (Hh) signaling pathway plays several diverse regulatory and patterning roles during organogenesis of the intestine and in the regulation of adult intestinal homeostasis. In the embryo, fetus, and adult, intestinal Hh signaling is paracrine: Hh ligands are expressed in the endodermally derived epithelium, while signal transduction is confined to the mesenchymal compartment, where at least a dozen distinct cell types are capable of responding to Hh signals. Epithelial Hh ligands not only regulate a variety of mesenchymal cell behaviors, but they also direct these mesenchymal cells to secrete additional soluble factors (e.g., Wnts, Bmps, inflammatory mediators) that feed back to regulate the epithelial cells themselves. Evolutionary conservation of the core Hh signaling pathway, as well as conservation of epithelial/mesenchymal cross talk in the intestine, has meant that work in many diverse model systems has contributed to our current understanding of the role of this pathway in intestinal organogenesis, which is reviewed here.
Expected final online publication date for the Annual Review of Physiology, Volume 83 is February 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Branching morphogenesis establishes the tree-like architecture of multiple organs during embryonic development. It requires coordinated, dynamic remodeling and signaling between tissues and the extracellular matrix (ECM). We review similarities and differences in the principles and mechanisms of three major examples of branching morphogenesis: lung, kidney, and salivary gland. These developing organs utilize interactions between embryonic epithelium, mesenchyme, and ECM, along with growth factor signaling, to generate distinct morphological architectures. The specific signaling molecules required and extent of stereotypic patterning of branches differ between these organs. Epithelial interactions with the basement membranes and other matrix proteins are also generally important, for example, for local remodeling to permit tissue expansion. Live-organ imaging and computational modeling are providing novel insights into the mechanical and signaling basis of branching morphogenesis. Combining multiple approaches will be necessary for deeper understanding of branching and to apply recent mechanistic insights to tissue engineering.
The centrosome, a major microtubule organizer, has important functions in regulating the cytoskeleton as well as the position of cellular structures and orientation of cells within tissues. The centrosome serves as the main cytoskeleton-organizing centre in the cell and is the classical site of microtubule nucleation and anchoring. For these reasons, centrosomes play a very important role in morphogenesis, not just in the early stages of cell divisions but also in the later stages of organogenesis. Many organs such as lung, kidney and blood vessels develop from epithelial tubes that branch into complex networks. Cells in the nervous system also form highly branched structures in order to build complex neuronal networks. During branching morphogenesis, cells have to rearrange within tissues though multicellular branching or through subcellular branching, also known as single-cell branching. For highly branched structures to be formed during embryonic development, the cytoskeleton needs to be extensively remodelled. The centrosome has been shown to play an important role during these events.
Neurons have evolved specialized growth structures to reach and innervate their target cells. These growth cones express specific receptor molecules that sense environmental cues and transform them into steering decisions. Historically, various concepts of axon guidance have been developed to better understand how axons reach and identify their targets. The essence of these efforts seems to be that growth cones require solid substrates and that major guidance decisions are initiated by extracellular cues. These sometimes highly conserved ligands and receptors have been extensively characterized and mediate four major guidance forces: chemoattraction, chemorepulsion, contact attraction and contact repulsion. However, during development, cells, too, do migrate in order to reach molecularly-defined niches at target locations. In fact, axonal growth could be regarded as a special case of cellular migration, where only a highly polarized portion of the cell is elongating. Here, I combine several examples from genetically tractable model organisms, such as Drosophila or zebrafish, in which cells and axons are guided by attractive cues. Regardless, if these cues are secreted into the extracellular space or exposed on cellular surfaces, migrating cells and axons seem to keep close contact with these attractants and seem to detect them right at their source. Migration towards and along such substrate-derived attractants seem to be particularly robust, as genetic deletion induces obvious searching behaviors and permanent guidance errors. In addition, forced expression of these factors in ectopic tissues is highly distractive too, regardless of the pattern of other endogenous cues. Thus, guidance and migration towards and along attractive tissues is a powerful steering mechanism that exploits affinity differences to the surroundings and, in some instances, determines growth trajectories from source to target region.
We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.
The development of air-filled respiratory organs
is crucial for survival at birth. We used a combination
of live imaging and genetic analysis to
dissect respiratory organ maturation in the
embryonic Drosophila trachea. We found that
tracheal tube maturation entails three precise
epithelial transitions. Initially, a secretion burst
deposits proteins into the lumen. Solid luminal
material is then rapidly cleared from the tubes,
and shortly thereafter liquid is removed. To
elucidate the cellular mechanisms behind these
transitions, we identified gas-filling-deficient
mutants showing narrow or protein-clogged
tubes. These mutations either disrupt endoplasmatic
reticulum-to-Golgi vesicle transport
or endocytosis. First, Sar1 is required for protein
secretion, luminal matrix assembly, and
diametric tube expansion. Subsequently, a
sharp pulse of Rab5-dependent endocytic
activity rapidly internalizes and clears luminal
contents. The coordination of luminal matrix
secretion and endocytosis may be a general
mechanism in tubular organ morphogenesis
and maturation.
Key Words organ development, respiratory system, epithelium, FGF, hypoxia s Abstract Many organs including the mammalian lung and vascular system con-sist of branched tubular networks that transport essential gases or fluids, but the genetic programs that control the development of these complex three-dimensional structures are not well understood. The Drosophila melanogaster tracheal (respiratory) system is a network of interconnected epithelial tubes that transports oxygen and other gases in the body and provides a paradigm of branching morphogenesis. It develops by se-quential sprouting of primary, secondary, and terminal branches from an epithelial sac of ∼80 cells in each body segment of the embryo. Mapping of the cell movements and shape changes during the sprouting process has revealed that distinct mechanisms of epithelial migration and tube formation are used at each stage of branching. Ge-netic dissection of the process has identified a general program in which a fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) are used repeatedly to control branch budding and outgrowth. At each stage of branching, the mechanisms controlling FGF expression and the downstream signal transduction pathway change, altering the pattern and structure of the branches that form. During terminal branching, FGF expression is regulated by hypoxia, ensuring that tracheal structure matches cel-lular oxygen need. A branch diversification program operates in parallel to the general budding program: Regional signals locally modify the general program, conferring specific structural features and other properties on individual branches, such as their substrate outgrowth preferences, differences in tube size and shape, and the ability to fuse to other branches to interconnect the network.
Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. Here, we have analyzed a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, we show that DAAM plays no essential role in planar cell polarity signaling, but it has striking requirements in organizing apical actin cables that define the taenidial fold pattern of the tracheal cuticle. These observations provide evidence the first time that the function of the taenidial organization is to prevent the collapse of the tracheal tubes. Our results indicate that although DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system.
The embryonic tracheal system in Drosophila develops from placodes of precursor cells on the ectoderm. A transcription factor of the basic helix-loop-helix (bHLH)-PAS family, which is expressed in the nuclei of the tracheal cells throughout development, was identified. The protein shows the highest degree of homology to the Single-minded (Sim) protein. The transcript represents the previously identified trachealess (trh) locus, essential for tracheal development. Ectopic expression of trh leads to generation of extra tracheal pits and branches and to the expression of tracheal markers by patches of ectodermal cells. The expression of trh is consistent with a biphasic mode of transcriptional regulation. Expression is first induced by exogenous cues and is subsequently autoregulated. trh is also expressed and required in the posterior spiracles and the salivary gland ducts. The role of Trachealess in the formation of several tubular tissues in the embryo suggests that it may induce a general fate of branched tubular structures of epithelial origin.
The tracheal (respiratory) system of Drosophila melanogaster is a branched network of epithelial tubes that ramifies throughout the body and transports oxygen to the tissues. It forms by a series of sequential branching events in each hemisegment from T2 to A8. Here we present a cellular and initial genetic analysis of the branching process. We show that although branching is sequential it is not iterative. The three levels of branching that we distinguish involve different cellular mechanisms of tube formation. Primary branches are multicellular tubes that arise by cell migration and intercalation; secondary branches are unicellular tubes formed by individual tracheal cells; terminal branches are subcellular tubes formed within long cytoplasmic extensions. Each level of branching is accompanied by expression of a different set of enhancer trap markers. These sets of markers are sequentially activated in progressively restricted domains and ultimately individual tracheal cells that are actively forming new branches. A clonal analysis demonstrates that branching fates are not assigned to tracheal cells until after cell division ceases and branching begins. We further show that the breathless FGF receptor, a tracheal gene required for primary branching, is also required to activate expression of markers involved in secondary branching and that the pointed ETS-domain transcription factor is required for secondary branching and also to activate expression of terminal branch markers. The combined morphological, marker expression and genetic data support a model in which successive branching events are mechanistically and genetically distinct but coupled through the action of a tracheal gene regulatory hierarchy.
We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.
During development of tubular networks such as the mammalian vascular system, the kidney and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous network. Little is known of the cellular mechanisms or molecular control of epithelial tube fusion. We describe the cellular dynamics of a tracheal fusion event in Drosophila and identify a gene regulatory hierarchy that controls this extraordinary process. A tracheal cell located at the developing fusion point expresses a sequence of specific markers as it grows out and contacts a similar cell from another tube; the two cells adhere and form an intercellular junction, and they become doughnut-shaped cells with the lumen passing through them. The early fusion marker Fusion-1 is identified as the escargot gene. It lies near the top of the regulatory hierarchy, activating the expression of later fusion markers and repressing genes that promote branching. Ectopic expression of escargot activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans. This establishes a simple genetic system to study fusion of epithelial tubes.
The molecular basis for patterning of complex organ structures like the lung and insect tracheal system is unknown. Here, we describe the Drosophila gene branchless (bnl) and demonstrate that it is a key determinant of the tracheal branching pattern. bnl is required for tracheal branching and is expressed dynamically in clusters of cells surrounding the developing tracheal system at each position where a new branch will form and grow out. Localized misexpression of bnl can direct branch formation and outgrowth to new positions. Generalized misexpression activates later programs of tracheal gene expression and branching, resulting in massive networks of branches. bnl encodes a homolog of mammalian fibroblast growth factors (FGFs) and appears to function as a ligand for the breathless receptor tyrosine kinase, an FGF receptor homolog expressed on developing tracheal cells. The results suggest that this FGF pathway specifies the tracheal branching pattern by guiding tracheal cell migration during primary branch formation and then activating later programs of finer branching at the ends of growing primary branches.
Coordination of cell motility and adhesion is essential for concerted movement of tissues during animal morphogenesis. The Drosophila tracheal network is formed by branching, migration and fusion of tubular ectodermal epithelia. Tracheal tip cells, located at the end of each branch that is going to fuse, extend filopodia to search for targets and later change their cell shape to a seamless ring to allow passage of lumen. The cell adhesion molecule DE-cadherin accumulates at the site of contact to form a ring that marks the site of lumen entry and is essential for the fusion. DE-cadherin expression in tip cells of a subset of branches is dependent on escargot, a zinc finger gene expressed in all tip cells. Such escargot mutant tip cells failed to adhere to each other and continued to search for alternative targets by extending long filopodia. We present evidence indicating escargot positively regulates transcription of the DE-cadherin gene, shotgun. Overexpression of DE-cadherin rescued the defect in one of the fusion points in escargot mutants, demonstrating an essential role of DE-cadherin in target recognition and identifying escargot as a key regulator of cell adhesion and motility in tracheal morphogenesis.
During mouse lung morphogenesis, the distal mesenchyme regulates the growth and branching of adjacent endoderm. We report here that fibroblast growth factor 10 (Fgf10) is expressed dynamically in the mesenchyme adjacent to the distal buds from the earliest stages of lung development. The temporal and spatial pattern of gene expression suggests that Fgf10 plays a role in directional outgrowth and possibly induction of epithelial buds, and that positive and negative regulators of Fgf10 are produced by the endoderm. In transgenic lungs overexpressing Shh in the endoderm, Fgf10 transcription is reduced, suggesting that high levels of SHH downregulate Fgf10.
Addition of FGF10 to embryonic day 11.5 lung tissue (endoderm plus mesenchyme) in Matrigel or collagen gel culture elicits a cyst-like expansion of the endoderm after 24 hours. In Matrigel, but not collagen, this is followed by extensive budding after 48-60 hours. This response involves an increase in the rate of endodermal cell proliferation. The activity of FGF1, FGF7 and FGF10 was also tested directly on isolated endoderm in Matrigel culture. Under these conditions, FGF1 elicits immediate endodermal budding, while FGF7 and FGF10 initially induce expansion of the endoderm. However, within 24 hours, samples treated with FGF10 give rise to multiple buds, while FGF7-treated endoderm never progresses to bud formation, at all concentrations of factor tested. Although exogenous FGF1, FGF7 and FGF10 have overlapping activities in vitro, their in vivo expression patterns are quite distinct in relation to early branching events. We conclude that, during early lung development, localized sources of FGF10 in the mesoderm regulate endoderm proliferation and bud outgrowth.
Antagonists of several growth factor signaling pathways play important roles in developmental patterning by limiting the range of the cognate inducer. Here, we describe an antagonist of FGF signaling that patterns apical branching of the Drosophila airways. In wild-type embryos, the Branchless FGF induces secondary branching by activating the Breathless FGF receptor near the tips of growing primary branches. In sprouty mutants, the FGF pathway is overactive and ectopic branches are induced on the stalks of primary branches. We show that FGF signaling induces sprouty expression in the nearby tip cells, and sprouty acts nonautonomously and in a competitive fashion to block signaling to the more distant stalk cells. sprouty encodes a novel cysteine-rich protein that defines a new family of putative signaling molecules that may similarly function as FGF antagonists in vertebrate development.
Fibroblast growth factor (FGF) signaling is required for normal epithelial branching in the respiratory system of several species. Recent studies have shown that FGF-10 may be a key regulator of lung branching morphogenesis, based on its pattern of expression in the early lung and its ability to induce epithelial budding in vitro. In this study we investigate whether FGF-10 is able to direct lung epithelial buds to proper positions during development . We maintained localized high levels of FGF-10 in cultured lungs using FGF-10-soaked heparin beads. FGF-10 exerts a powerful chemoattractant effect on the distal but not on proximal lung epithelium. Epithelial buds grow toward an FGF-10 source within 24 h, and subsequently form concentric layers of epithelium around the bead. BrdU incorporation analysis suggests that FGF-10, in contrast to FGF-7, is a modest proliferation factor for the lung epithelium. In the absence of mesenchyme FGF-10 requires an associated proliferative signal to induce bud migration. This can be provided by extract from lung mesenchyme, or by FGF-7, a growth factor also present in the early embryonic lung. FGF-10 does not seem to interfere with early epithelial cell differentiation. The chemoattractant effect of FGF-10 in the lung epithelium is reminiscent of the patterning effect of the Drosophila FGF ortholog branchless in the developing tracheal epithelium, suggesting that the function of these genes has been conserved during evolution.
Fibroblast growth factors (FGFs) bind to FGF receptors, transmembrane tyrosine kinases that activate mitogenic, motogenic, and differentiative responses in different tissues. While there has been substantial progress in elucidating the Ras-MAP kinase pathway that mediates the differentiative responses, the signal transduction pathways that lead to directed cell migrations are not well defined. Here we describe a Drosophila gene called stumps that is required for FGF-dependent migrations of tracheal and mesodermal cells. These migrations are controlled by different FGF ligands and receptors, and they occur by different cellular mechanisms: the tracheal migrations occur as part of an epithelium whereas the mesodermal migrations are fibroblast-like. In the stumps mutant, tracheal cells fail to move out from the epithelial sacs, and only rudimentary tracheal branches form. Mesodermal cells fail in their dorsal migrations after gastrulation. The stumps mutation does not block all FGF signaling effects in these tissues: both random cell migrations and Ras-MAP kinase-mediated induction of FGF-specific effector genes occurred upon ectopic expression of the ligand or upon expression of a constitutively activated Ras protein in the migrating cells. The results suggest that stumps function promotes FGF-directed cell migrations, either by potentiating the FGF signaling process or by coupling the signal to the cellular machinery required for directed cell movement.
The Drosophila tracheal system consists of a stereotyped network of epithelial tubes formed by several tracheal cell types. By the end of embryogenesis, when the general branching pattern is established, some specialised tracheal cells then mediate branch fusion while others extend fine terminal branches. Here evidence is presented that the Notch signalling pathway acts directly in the tracheal cells to distinguish individual fates within groups of equivalent cells. Notch helps to single out those tracheal cells that mediate branch fusion by blocking their neighbours from adopting the same fate. This function of Notch would require the restricted activation of the pathway in specific cells. In addition, and probably later, Notch also acts in the selection of those tracheal cells that extend the terminal branches. Both the localised expression and the mutant phenotypes of Delta, a known ligand for Notch, suggest that Delta may activate Notch to specify cell fates at the tips of the developing tracheal branches.
The fibroblast growth factor receptor 2 gene is differentially spliced to encode two transmembrane tyrosine kinase receptor proteins that have different ligand-binding specificities and exclusive tissue distributions. We have used Cre-mediated excision to generate mice lacking the IIIb form of fibroblast growth factor receptor 2 whilst retaining expression of the IIIc form. Fibroblast growth factor receptor 2(IIIb) null mice are viable until birth, but have severe defects of the limbs, lung and anterior pituitary gland. The development of these structures appears to initiate, but then fails with the tissues undergoing extensive apoptosis. There are also developmental abnormalities of the salivary glands, inner ear, teeth and skin, as well as minor defects in skull formation. Our findings point to a key role for fibroblast growth factor receptor 2(IIIb) in mesenchymal-epithelial signalling during early organogenesis.
The proper size of epithelial tubes is critical for the function of the lung, kidney, vascular system and other organs, but the genetic and cellular mechanisms that control epithelial tube size are unknown. We investigated tube size control in the embryonic and larval tracheal (respiratory) system of Drosophila. A morphometric analysis showed that primary tracheal branches have characteristic sizes that undergo programmed changes during development. Branches grow at different rates and their diameters and lengths are regulated independently: tube length increases gradually throughout development, whereas tube diameter increases abruptly at discrete times in development. Cellular analysis and manipulation of tracheal cell number using cell-cycle mutations demonstrated that tube size is not dictated by the specific number or shape of the tracheal cells that constitute it. Rather, tube size appears to be controlled by coordinately regulating the apical (lumenal) surface of tracheal cells. Genetic analysis showed that tube sizes are specified early by branch identity genes, and the subsequent enlargement of branches to their mature sizes and maintenance of the expanded tubes involves a new set of genes described here, which we call tube expansion genes. This work establishes a genetic system for investigating tube size regulation, and provides an outline of the genetic program and cellular events underlying tracheal tube size control.
Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path.
We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues.
We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos.
Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.
Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. We used the tracheal system of Drosophila to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. We found that a reduced Rac activity led to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibited dynamic epithelial cell rearrangement. Hyperactivation of Rac, on the other hand, inhibited assembly of newly synthesized E-Cadherin into cell junctions and caused loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules.
The formation of branched epithelial networks is fundamental to the development of many organs, such as the lung, the kidney or the vasculature. Little is known about the mechanisms that control cell rearrangements during tubulogenesis and regulate the size of individual tubes. Recent studies indicate that whereas the basal surface of tube cells interacts with the surrounding tissues and helps to shape the ramification pattern of tubular organs, the apical surface has an important role in the regulation of tube diameter and tube growth. Here we report that two proteins, Piopio (Pio) and Dumpy (Dp), containing a zona pellucida (ZP) domain are essential for the generation of the interconnected tracheal network in Drosophila melanogaster. Pio is secreted apically, accumulates in the tracheal lumen and possibly interacts with Dp through the ZP domains. In the absence of Pio and Dp, multicellular tubes do not rearrange through cell elongation and cell intercalation to form narrow tubes with autocellular junctions; instead they are transformed into multicellular cysts, which leads to a severe disruption of the branched pattern. We propose that an extracellular matrix containing Pio and Dp provides a structural network in the luminal space, around which cell rearrangements can take place in an ordered fashion without losing interconnections. Our results suggest that a similar structural role might be attributed to other ZP-domain proteins in the formation of different branched organs.
Human nm23 has been implicated in suppression of metastasis in various cancers, but the underlying mechanism of such activity has not been fully understood. Using Drosophila tracheal system as a genetic model, we examined the function of the Drosophila homolog of nm23, the awd gene, in cell migration. We show that loss of Drosophila awd results in dysregulated tracheal cell motility. This phenotype can be suppressed by reducing the dosage of the chemotactic FGF receptor (FGFR) homolog, breathless (btl), indicating that btl and awd are functionally antagonists. In addition, mutants of shi/dynamin show similar tracheal phenotypes as in awd and exacerbate those in awd mutant, suggesting defects in vesicle-mediated turnover of FGFR in the awd mutant. Consistent with this, Btl-GFP chimera expressed from a cognate btl promoter-driven system accumulate at high levels on tracheal cell membrane of awd mutants as well as in awd RNA duplex-treated cultured cells. Thus, we propose that awd regulates tracheal cell motility by modulating the FGFR levels, through a dynamin-mediated pathway.
Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in
Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration.
We have performed a functional characterization of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically
in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, we demonstrate that Dof is a specific substrate
for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, we identify two regions important for Dof function in mesodermal and
tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon
receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment
represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, our results
also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal
cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response.
Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.
The fibroblast growth factor receptor 2 gene is differentially spliced to encode two transmembrane tyrosine kinase receptor proteins that have different ligand-binding specificities and exclusive tissue distributions. We have used Cre-mediated excision to generate mice lacking the IIIb form of fibroblast growth factor receptor 2 whilst retaining expression of the IIIc form. Fibroblast growth factor receptor 2(IIIb) null mice are viable until birth, but have severe defects of the limbs, lung and anterior pituitary gland. The development of these structures appears to initiate, but then fails with the tissues undergoing extensive apoptosis. There are also developmental abnormalities of the salivary glands, inner ear, teeth and skin, as well as minor defects in skull formation. Our findings point to a key role for fibroblast growth factor receptor 2(IIIb) in mesenchymal-epithelial signalling during early organogenesis.
The generation of bodies and body parts with specific shapes and sizes has been a longstanding issue in biology. Morphogenesis in general and organogenesis in particular are complex events that involve global changes in cell populations in terms of their proliferation, migration, differentiation and shape. Recent studies have begun to address how these synchronized changes are controlled by the genes that specify cell fate and by the ability of cells to respond to extracellular cues. In particular, a notable shift in this research has occurred owing to the ability to address these issues in the context of the whole organism. For such studies, the Drosophila tracheal system has proven to be a particularly appropriate model. Here, my aim is to highlight some ideas that have arisen through our studies, and those from other groups, of Drosophila tracheal development. Rather than providing an objective review of the features of tracheal development, I intend to discuss some selected notions that I think are relevant to the question of shape generation.
A Drosophila homolog of the fibroblast growth factor (FGF) receptor was isolated and structurally characterized. After EMS mutagenesis or imprecise excisions of marked P elements inserted upstream to the gene, a phenotypic series of mutations in the locus was isolated. The mutants exhibit defects in the two embryonic tissues in which the receptor is expressed: the tracheal system and the midline. The tracheal cells fail to migrate in severe mutants and remain within the tracheal pits. Hypomorphic alleles exhibit partial migration of all tracheal branches; thus, the locus was termed breathless (btl). In the midline of the mutant embryos, the posterior pair of midline glial cells begins to migrate anteriorly, but fails to reach the posterior commissure. Abnormalities in cell migration appear to be a common denominator for the btl defects in these two disparate tissues. In hypomorphic mutants the cells exhibit partial migration but follow the normal tracts, suggesting that the presence of this receptor is essential for the ability of the migrating cells to recognize external guiding cues.
We report that the region-specific homeotic gene spalt affects the Drosophila tracheal system at two different stages of embryonic development. Both lack-of-function and gain-of-function experiments show that blastodermal spalt activity restricts tracheal development to 10 bilaterally positioned pairs of tracheal placodes in the trunk region by repressing placode formation in parasegments 2, 3 and 14. The results suggest that the activity of the zinc-finger type transcription factor encoded by spalt suppresses the molecular pathway that establishes tracheal development. spalt function is also necessary for the directed migration of the dorsal trunk cells, a distinct subset of tracheal cells. This process is a prerequisite for the formation of the dorsal trunk generated by fusion of adjacent tracheal metameres into a common tubular structure. The directed cell migration, in which spalt gene function participates, seems to be independent of branch fusion and general tracheal cell migration processes.
Cell proliferation is an essential requirement for epithelial expansion and tubular branching; however, little is known of how these events are coupled during morphogenesis. We have previously shown that, in the absence of mesenchyme, fibroblast growth factor 1 (FGF-1) elicits budding of the mouse lung epithelium cultured in a basement membrane matrix. Although bud formation seems to be the manifestation of a localized response of lung epithelial cells to FGF-1, it is unclear whether budding results from induction of differential rates of cell proliferation within the epithelium. We performed continuous labeling and pulse-chase experiments in FGF-1-treated mesenchyme-free lung epithelial cultures at distinct stages of bud induction using bromodeoxyuridine (BrdU), to determine when and to what extent cell proliferation contributes to bud formation. When explants were incubated with BrdU either before bud induction (0-18 hr in culture) or at the onset of budding (24-30 hr), labeled nuclei were found distributed throughout the entire explant. In contrast, BrdU incubation after the onset of budding (30-48 hr) resulted in labeling concentrated in the budding areas, and a decrease of labeling toward the proximal region of the explant, between buds. These results demonstrate that differential rates of cell proliferation between bud and nonbud areas do not appear until when buds are almost completely formed. Thus, in the developing lung epithelium in vitro, bud outgrowth is not triggered by induction of localized cell proliferation.
Receptor tyrosine kinases (RTKs) transmit signals to the cell nucleus via the MAP kinase (MAPK) cascade, using specific molecules to link the activated receptors to the MAPK cascade activator, Ras. We have identified a component of the FGF receptor (FGFR) signal transduction pathway, Downstream of FGFR (Dof). Dof is an intracellular protein that is essential for signal transmission by the FGFR and acts downstream of the receptor and upstream of Ras. Unlike other signaling molecules that act downstream of RTKs, Dof is not expressed ubiquitously but is present exclusively in cells that express FGFRs. Dof is needed in these cells for activation of the MAPK cascade via FGF signaling, but not for activation via other RTK ligands. Dof therefore appears to be committed exclusively to FGFR-mediated signal transduction.
The interactions between fibroblast growth factors (FGF) and their receptors have important roles in mediating mesenchymal-epithelial cell interactions during embryogenesis. In particular, Fgf10 is predicted to function as a regulator of brain, lung and limb development on the basis of its spatiotemporal expression pattern in the developing embryo. To define the role of Fgf10, we generated Fgf10-deficient mice. Fgf10-/- mice died at birth due to the lack of lung development. Trachea was formed, but subsequent pulmonary branching morphogenesis was disrupted. In addition, mutant mice had complete truncation of the fore- and hindlimbs. In Fgf10-/- embryos, limb bud formation was initiated but outgrowth of the limb buds did not occur; however, formation of the clavicles was not affected. Analysis of the expression of marker genes in the mutant limb buds indicated that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) did not form. Thus, we show here that Fgf10 serves as an essential regulator of lung and limb formation.
Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. We report that Dpp signaling is also required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling is Delta. In conditional Notch loss of function mutants additional tracheal cells adopt the fusion cell fate and ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch(ts1) mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches.
The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless that activates a receptor tyrosine kinase Breathless and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tip of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Here we show that Notch signaling plays crucial roles in the singling out process of the fusion cell. We found that Notch is activated in tracheal cells by Branchless signaling through stimulation of &Dgr; expression at the tip of tracheal branches and that activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tip of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis.
The Drosophila tracheal (respiratory) system is a tubular epithelial network that delivers oxygen to internal tissues. Sprouting of the major tracheal branches is stereotyped and controlled by hard-wired developmental cues. Here we show that ramification of the fine terminal branches is variable and regulated by oxygen, and that this process is controlled by a local signal or signals produced by oxygen-starved cells. We provide evidence that the critical signal is Branchless (Bnl) FGF, the same growth factor that patterns the major branches during embryogenesis. During larval life, oxygen deprivation stimulates expression of Bnl, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. Thus, a single growth factor is reiteratively used to pattern each level of airway branching, and the change in branch patterning results from a switch from developmental to physiological control of its expression.
The Drosophila tracheal system is a branched tubular structure that supplies air to target tissues. The elaborate tracheal morphology is shaped by two linked inductive processes, one involving the choice of cell fates, and the other a guided cell migration. We will describe the molecular basis for these processes, and the allocation of cell fate decisions to four temporal hierarchies. First, tracheal placodes are specified within the embryonic ectoderm. Subsequently, branch fates are allocated within the tracheal placodes, prior to migration. Localized presentation of the FGF ligand, Branchless, to tracheal cells that express the FGF receptor, Breathless, guides migration. Once cell migration is initiated, distinct cell fates are determined within each migrating branch. Finally, inhibitory feedback mechanisms ensure the correct assignment of these fates. Tracheal cell fate choices are determined by signaling cascades triggered by signals emanating from the tracheal cells, as well as by ligands produced by adjacent tissues.
Growth and morphogenesis during development depend both on patterning genes, which assign positional information, and on genes that regulate mechanical forces. The dumpy gene of the fruit fly Drosophila melanogaster is an example of the latter class, with mutant phenotypes affecting size and shape of the limbs, thoracic cuticle, trachea and mouthparts.
The genetically complex dumpy locus was found to span over 100 kb and encode a gigantic 2.5 MDa extracellular matrix protein. Dumpy represents an extreme form of modular protein evolution, containing 308 epidermal growth factor (EGF) modules, interspersed with a new module class, DPY, and terminating in a crosslinking zona pellucida domain and membrane anchor sequence. We determined the three-dimensional structure of the DPY module by nuclear magnetic resonance (NMR) spectroscopy and found that it forms a disulphide-stabilised beta sheet motif, capable of linking end-to-end with EGF modules to form a fibre. Consistent with its cuticle phenotypes, dumpy is expressed at several sites of cuticle-epidermal cell attachment, including the trachea and the muscle tendon cells, which mediate anchorage of the muscles to the cuticle.
The dumpy gene encodes a gigantic extracellular molecule that we predict to be a membrane-anchored fibre of almost a micrometer in length. Insertion and crosslinking of this fibre within the cuticle may provide a strong anchor for the underlying tissue, allowing it to maintain mechanical tension at sites under stress. This would explain its contribution to tissue morphogenesis through its regulation of mechanical properties.
Branching morphogenesis is a widely used strategy to increase the surface area of a given organ. A number of tissues undergo branching morphogenesis during development, including the lung, kidney, vascular system and numerous glands. Until recently, very little has been known about the genetic principles underlying the branching process and about the molecules participating in organ specification and branch formation. The tracheal system of insects represents one of the best-characterised branched organs. The tracheal network provides air to most tissues and its development during embryogenesis has been studied intensively at the morphological and genetic level. More than 30 genes have been identified and ordered into sequential steps controlling branching morphogenesis. These studies have revealed a number of important principles that might be conserved in other systems.
Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short Stop (shot). In these fusion cells, the apical surface determinant Discs Lost (Dlt) is subsequently deposited and new lumen forms along the track. In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led us to investigate whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. We therefore propose that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization.
In the developing tracheal system of Drosophila melanogaster, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic (Dpp) signaling is strictly required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, we find that the two signaling systems affect different cellular functions required for branching morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth.
As organisms have evolved in size and complexity, tubular systems have developed to enable the efficient transport of substances into and out of tissues. These tubular systems are generated using strategies that are based on common elements of cell behaviour, including cell polarization, tube migration to target sites, cell-fate diversification and localization of specialized cells to different regions of the tube system. Using examples from both invertebrate and vertebrate systems, this review highlights progress in understanding these basic principles and briefly discusses the possible evolution of strategies to regulate the morphogenesis of tubular systems.
In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.
The Drosophila adult has a complex tracheal system that forms during the pupal period. We have studied the derivation of part of this system, the air sacs of the dorsal thorax. During the third larval instar, air sac precursor cells bud from a tracheal branch in response to FGF, and then they proliferate and migrate to the adepithelial layer of the wing imaginal disc. In addition, FGF induces these air sac precursors to extend cytoneme-like filopodia to FGF-expressing cells. These findings provide evidence that FGF is a mitogen in Drosophila, correlate growth factor signaling with filopodial contact between signaling and responding cells, and suggest that FGF can act on differentiated tracheal cells to induce a novel behavior and role.
The zebrafish has recently emerged as an advantageous model organism for studying how the stereotypic and evolutionarily conserved network of vertebrate blood vessels arises during development. The ability to screen for vascular-specific mutants and to image and experimentally manipulate blood vessels throughout living embryos has already yielded new insights into the anatomy of the early vasculature, the dynamics of growing blood vessels, the specification of early vascular progenitors, and arterial-venous differentiation of blood vessels.
Branching morphogenesis involves the restructuring of epithelial tissues into complex and organized ramified tubular networks. Early rounds of branching are controlled genetically in a hardwired fashion in many organs, whereas later, branching is stochastic, responding to environmental cues. We discuss this sequential process from formation of an organ anlage and invagination of the epithelium to branch initiation and outgrowth in several model systems including Drosophila trachea and mammalian lung, mammary gland, and kidney.
Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. The biological effects of VEGF are mediated by two receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2, which differ considerably in signaling properties. Non-signaling co-receptors also modulate VEGF RTK signaling. Currently, several VEGF inhibitors are undergoing clinical testing in several malignancies. VEGF inhibition is also being tested as a strategy for the prevention of angiogenesis, vascular leakage and visual loss in age-related macular degeneration.
Blood vessels constitute the first organ in the embryo and form the largest network in our body but, sadly, are also often deadly. When dysregulated, the formation of new blood vessels contributes to numerous malignant, ischemic, inflammatory, infectious and immune disorders. Molecular insights into these processes are being generated at a rapidly increasing pace, offering new therapeutic opportunities that are currently being evaluated.
The cellular architecture of tubular organs suggests striking similarities in the mechanisms of tubulogenesis between species. The formation of the Drosophila respiratory organ (trachea) highlights the basic principles of branch patterning and tube growth that generate a highly elaborate but stereotyped epithelial tubular network. Oriented cell migration, changes in cell shape, selective growth of the apical cell membrane and intracellular lumen formation are essential events in this process. These morphogenetic processes build four structurally distinct classes of tubes that facilitate optimal airflow and gas exchange with target tissues. The molecular players in these plots include attractant and repellent signals, differentiation factors that cause a high diversity of cell fates within the epithelium, and determinants of tube formation and dimensions.
During tracheal development in Drosophila, some branches join to form a continuous luminal network. Specialized cells at the branch tip, called fusion cells, extend filopodia to make contact and become doughnut shaped to allow passage of the lumen. These morphogenetic processes accompany the highly regulated cytoskeletal reorganization of fusion cells. We identified the Drosophila formin3 (form3) gene that encodes a novel formin and plays a role in tracheal fusion. Formins are a family of proteins characterized by highly conserved formin homology (FH) domains. The formin family functions in various actin-based processes, including cytokinesis and cell polarity. During embryogenesis, form3 mRNA is expressed mainly in the tracheal system. In form3 mutant embryos, the tracheal fusion does not occur at some points. This phenotype is rescued by the forced expression of form3 in the trachea. We used live imaging of GFP-moesin during tracheal fusion to show that an F-actin structure that spans the adjoining fusion cells and mediates the luminal connection does not form at abnormal anastomosis sites in form3 mutants. These results suggested that Form3 plays a role in the F-actin assembly, which is essential for cellular rearrangement during tracheal fusion.
Blood vessels and nerves are complex, branched structures that share a high degree of anatomical similarity. Guidance of vessels and nerves has to be exquisitely regulated to ensure proper wiring of both systems. Several regulators of axon guidance have been identified and some of these are also expressed in endothelial cells; however, the extent to which their guidance functions are conserved in the vascular system is still incompletely understood. We show here that the repulsive netrin receptor UNC5B is expressed by endothelial tip cells of the vascular system. Disruption of the Unc5b gene in mice, or of Unc5b or netrin-1a in zebrafish, leads to aberrant extension of endothelial tip cell filopodia, excessive vessel branching and abnormal navigation. Netrin-1 causes endothelial filopodial retraction, but only when UNC5B is present. Thus, UNC5B functions as a repulsive netrin receptor in endothelial cells controlling morphogenesis of the vascular system.
This chapter discusses the imaging blood vessels in the Zebrafish. Recent evidence suggests that the genetic factors are critical in the formation of major vessels formed during early development. Understanding the mechanisms behind the emergence of these early vascular networks requires the use of genetically and experimentally accessible model vertebrates. The accessibility and optical clarity of the zebrafish embryo, and larva makes it particularly useful for studies of vascular development. Moreover, studies of developing vessels are likely to have far-reaching implications for human health, since understanding the mechanisms underlying the growth and morphogenesis of blood vessels has become critical for a number of important emerging clinical applications. Furthermore, proangiogenic and antiangiogenic therapies show great promise for treating ischemia and cancer, respectively, and a great deal of evort is going into uncovering and characterizing the factors that can be used to promote or inhibit vessel growth in vivo. Since many of the molecules that play key roles in developing vessels carry out analogous functions during postnatal angiogenesis, it seems likely that the zebrafish yield many important clinically applicable insights in the future.