Genetic control of branching morphogenesis during Drosophila tracheal development

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.     

The hindsight gene is required for epithelial maintenance and differentiation of the tracheal system in Drosophila

During animal development, morphogenesis of tissues and organs requires dynamic cell shape changes and movements that are accomplished without loss of epithelial integrity. Data from vertebrate and invertebrate systems have implicated several cell surface and cytoskeleton-associated molecules in the establishment and maintenance of epithelial architecture, but there has been little analysis of the genetic regulatory hierarchies that control epithelial morphogenesis in specific tissues. Here we show that the Drosophila Hindsight nuclear zinc-finger protein is required during tracheal morphogenesis for the maintenance of epithelial integrity and assembly of apical extracellular structures known as taenidia. In hindsight (hnt) mutants tracheal placodes form, invaginate, and undergo primary branching as well as early fusion events. Starting at midembryogenesis, however, the tracheal epithelium collapses or expands to give rise to sacs of tissue. While a subset of hnt mutant tracheal cells enters the apoptotic pathway, genetic suppression of apoptosis indicates that this is not the cause of the epithelial defects. Surviving hnt mutant tracheal cells retain cell-cell junctions and a normal subcellular distribution of apical markers such as Crumbs and DE-Cadherin. However, taenidia do not form on the lumenal surface of tracheal cells. While loss of epithelial integrity is a common feature of crumbs, stardust, and hnt mutants, defective assembly of taenidia is unique to hnt mutants. These data suggest that HNT is a tissue-specific factor that regulates maintenance of the tracheal epithelium as well as differentiation of taenidia.     

Tracheal development in Drosophila melanogaster as a model system for studying the development of a branched organ

The development of the tracheal system of Drosophila melanogaster represents a paradigm for studying the molecular mechanisms involved in the formation of a branched tubular network. Tracheogenesis has been characterized at the morphological, cellular and genetic level and a series of successive, but linked events have been described as the basis for the formation of the complex network of tubules which extend over the entire organism. Tracheal cells stop to divide early in the process of tracheogenesis and the formation of the interconnected network requires highly controlled cell migration events and cell shape changes. A number of genes involved in these two processes have been identified but in order to obtain a more complete view of branching morphogenesis, many more genes carrying essential functions have to be isolated and characterized. Here, we provide a progress report on our attempts to identify further genes expressed in the tracheal system. We show that empty spiracles (ems), a head gap gene, is required for the formation of a specific tracheal branch, the visceral branch. We also identified a Sulfotransferase and a Multiple Inositol Polyphosphate phosphatase that are strongly upregulated in tracheal cells and discuss their possible involvement in tracheal development.     

Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila

Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.     

The alternative migratory pathways of the Drosophila tracheal cells are associated with distinct subsets of mesodermal cells

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. Here we analyze how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. We find that cells in different branches exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. We also find that alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and propose a model for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints.     

Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions

The mechanisms by which the branching of epithelial tissue occurs and is regulated to generate different organ structures are not well understood. In this work, image analyses of the organ rudiments demonstrate specific epithelial branching patterns for the early lung and kidney; the lung type typically generating several side branches, whereas kidney branching was mainly dichotomous. Parameters such as the number of epithelial tips, the angle of the first branch, the position index of the first branch (PIFB) in a module, and the percentage of epithelial module type (PMT) were analysed. The branching patterns in the cultured lung and kidney, and in homotypic tissue recombinants recapitulated their early in vivo branching patterns. The parameters were applied to heterotypic tissue recombinants between lung mesenchyme and ureteric bud, and tip number, PIFB and PMT values qualified the change in ureter morphogenesis and the reprogramming of the ureteric bud with lung mesenchyme. All the values for the heterotypic recombinant between ureteric bud and lung mesenchyme were significantly different from those for kidney samples but similar to those of the lung samples. Hence, lung mesenchyme can instruct the ureteric bud to undergo aspects of early lung-type epithelial morphogenesis. Different areas of the lung mesenchyme, except the tracheal region, were sufficient to promote ureteric bud growth and branching. In conclusion, our findings provide morphogenetic parameters for monitoring epithelial development in early embryonic lung and kidney and demonstrate the use of heterotypic tissue recombinants as a model for studying tissue-specific epithelial branching during organogenesis.     

Multiple roles and interactions of tbx4 and tbx5 in development of the respiratory system

Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.     

Tracheal branching morphogenesis in Drosophila: new insights into cell behaviour and organ architecture

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.     

Real-time analysis of ureteric bud branching morphogenesis in vitro

While it is clear that the normal branching morphogenesis of the ureteric bud (UB) is critical for development of the metanephric kidney, the specific patterns of branching and growth have heretofore only been inferred from static images. Here, we present a systematic time-lapse analysis of UB branching morphogenesis during the early development of the mouse kidney in organ culture. Metanephric primordia from Hoxb7/GFP transgenic embryos were cultured for 3-4 days, and GFP images of the UB taken every 30 min were assembled into movies. Analysis of these movies (available as )revealed that the UB is a highly plastic structure, which can branch in a variety of complex patterns, including terminal bifid, terminal trifid, and lateral branching. To examine kinetic parameters of branching and elongation, skeletal representations of the UB were used to measure the number of segments and branch points and the length of each segment as a function of time and of branch generation. These measurements provide a baseline for future studies on mutant kidneys with defects in renal development. To illustrate how these quantitative methods can be applied to the analysis of abnormal kidney development, we examined the effects of the MEK1 inhibitor PD98059 on renal organ cultures and confirmed a previous report that the drug has a specific inhibitory effect on UB branching as opposed to elongation.     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.     

NRAGE: A potential rheostat during branching morphogenesis

Branching morphogenesis is a developmental process characteristic of many organ systems. Specifically, during renal branching morphogenesis, its been postulated that the final number of nephrons formed is one key clinical factor in the development of hypertension in adulthood. As it has been established that BMPs regulate, in part, renal activity of p38 MAP kinase (p38^MAPK) and it has demonstrated that the cytoplasmic protein Neurotrophin Receptor MAGE homologue (NRAGE) augments p38^MAPK activation, it was hypothesized that a decrease in the expression of NRAGE during renal branching would result in decreased branching of the UB that correlated with changes in p38^MAPK activation. To verify this, the expression of NRAGE was reduced in ex vivo kidney explants cultures using antisense morpholino. Morpholino treated ex vivo kidney explants expression were severely stunted in branching, a trait that was rescued with the addition of exogenous GDNF. Renal explants also demonstrated a precipitous drop in p38^MAPK activation that too was reversed in the presence of recombinant GDNF. RNA profiling of NRAGE diminished ex vivo kidney explants resulted in altered expression of GDNF, Ret, BMP7 and BMPRIb mRNAs. Our results suggested that in early kidney development NRAGE might have multiple roles during renal branching morphogenesis through association with both the BMP and GDNF signaling pathways.     

Research Progress and Application of Plant Branching

Plant branching development plays an important role in plant morphogenesis (aboveground plant type), the number and angle of branches are important agronomic characters that determine crop plant type. Effective branches determine the number of panicles or pods of crops and then control the yield of crops. With the rapid development of plant genomics and molecular genetics, great progress has been made in the study of branching development. In recent years, a series of important branching-related genes have been validated from Arabidopsis thaliana, rice, pea, tomato and maize mutants. It is reviewed that plant branching development is controlled by genetic elements and plant hormones, such as auxin, cytokinin and lactones (or lactone derivatives), as well as by environment and genetic elements. Meanwhile, shoot architecture in crop breeding was discussed in order to provide theoretical basis for the study of crop branching regulation.     

The Fog signaling pathway: Insights into signaling in morphogenesis

Epithelia form the building blocks of many tissue and organ types. Epithelial cells often form a contiguous 2-dimensional sheet that is held together by strong adhesions. The mechanical properties conferred by these adhesions allow the cells to undergo dramatic three-dimensional morphogenetic movements while maintaining cell–cell contacts during embryogenesis and post-embryonic development. The Drosophila Folded gastrulation pathway triggers epithelial cell shape changes that drive gastrulation and tissue folding and is one of the most extensively studied examples of epithelial morphogenesis. This pathway has yielded key insights into the signaling mechanisms and cellular machinery involved in epithelial remodeling. In this review, we discuss principles of morphogenesis and signaling that have been discovered through genetic and cell biological examination of this pathway. We also consider various regulatory mechanisms and the system?s relevance to mammalian development. We propose future directions that will continue to broaden our knowledge of morphogenesis across taxa.     

Renal branching morphogenesis: concepts, questions, and recent advances

The ureteric bud (UB) is an outgrowth of the Wolffian duct, which undergoes a complex process of growth, branching, and remodeling, to eventually give rise to the entire urinary collecting system during kidney development. Understanding the mechanisms that control this process is a fascinating problem in basic developmental biology, and also has considerable medical significance. Over the past decade, there has been significant progress in our understanding of renal branching morphogenesis and its regulation, and this review focuses on several areas in which there have been recent advances. The first section deals with the normal process of UB branching morphogenesis, and methods that have been developed to better observe and describe it. The next section discusses a number of experimental methodologies, both established and novel, that make kidney development in the mouse a powerful and attractive experimental system. The third section discusses some of the cellular processes that are likely to underlie UB branching morphogenesis, as well as recent data on cell lineages within the growing UB. The fourth section summarizes our understanding of the roles of two groups of growth factors that appear to be particularly important for the regulation of UB outgrowth and branching: GDNF and FGFs, which stimulate this process via tyrosine kinase receptors, and members of the TGFbeta family, including BMP4 and Activin A, which generally inhibit UB formation and branching.     

The role of cell proliferation and cellular shape change in branching morphogenesis of the embryonic mouse lung: analysis using aphidicolin and cytochalasins

The formation of induced supernumerary buds in the embryonic mouse tracheal epithelium has been used as a model system to analyse the respective roles of cell proliferation and microfilament-mediated cell shape change during branching morphogenesis. In order to analyse the mitotic events associated with the formation of epithelial buds, the induction of supernumerary tracheal buds by mesenchymal grafts was carried out with the inhibitor of DNA synthesis, aphidicolin, present in the culture medium for varying intervals of time during the 16-hour inductive process. The presence of aphidicolin for 10 to 16 hours of the inductive period blocks the formation of induced tracheal buds, whereas the presence of the inhibitor for half of that time (either the first 8 hours or the last 8 hours) does not prevent this morphogenetic event from taking place, although smaller buds resulted from induction under these conditions. Both the inhibition of DNA synthesis and the recovery from 10 microM aphidicolin treatment, as measured by 3H-thymidine incorporation, were found to occur rapidly. The addition of 2 microM dihydrocytochalasin B (or cytochalasin B) together with aphidicolin during the second half of the inductive period inhibits the formation of supernumerary buds and upon removal of the cytochalasin rapid formation of buds takes place. We conclude that the formation of epithelial buds during branching morphogenesis occurs as a result of enhanced localized cell proliferation coupled with epithelial cell shape change (or preservation of cell morphology) mediated by microfilaments, which have been observed in both the apical and basal cytoplasm of the epithelial cells in the region where branching of the trachea is taking place.