An Ichor-dependent apical extracellular matrix regulates seamless tube shape and integrity.

During sprouting angiogenesis in the vertebrate vascular system, and primary branching in the Drosophila tracheal system, specialized tip cells direct branch outgrowth and network formation. When tip cells lumenize, they form subcellular (seamless) tubes. How these seamless tubes are made, shaped and maintained remains poorly understood. Here we characterize a Drosophila mutant called ichor (ich), and show that ich is essential for the integrity and shape of seamless tubes in tracheal terminal cells. We find that Ich regulates seamless tubulogenesis via its role in promoting the formation of a mature apical extracellular matrix (aECM) lining the lumen of the seamless tubes. We determined that ich encodes a zinc finger protein (CG11966) that acts, as a transcriptional activator required for the expression of multiple aECM factors, including a novel membrane-anchored trypsin protease (CG8213). Thus, the integrity and shape of seamless tubes are regulated by the aECM that lines their lumens.

Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment.

Cellular tubes have diverse morphologies, including multicellular, unicellular and subcellular architectures. Subcellular tubes are found prominently within the vertebrate vasculature, the insect breathing system and the nematode excretory apparatus, but how such tubes form is poorly understood. To characterize the cellular mechanisms of subcellular tube formation, we have refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using our methods, we have found that subcellular tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. We have also developed correlative light/TEM procedures to identify labeled cells in TEM-fixed larval samples. Using this technique, we have found that Vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, our ultrastructural analysis methods could be useful for a wide range of cellular investigations in Drosophila larvae.

Exocyst-mediated membrane trafficking is required for branch outgrowth in Drosophila tracheal terminal cells.

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increase surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs at the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find that the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.

Drosophila mutants show NMD pathway activity is reduced, but not eliminated, in the absence of Smg6.

The nonsense-mediated mRNA decay (NMD) pathway is best known for targeting mutant mRNAs containing premature termination codons for rapid degradation, but it is also required for regulation of many endogenous transcripts. Components of the NMD pathway were originally identified by forward genetic screens in yeast and Caenorhabditis elegans. In other organisms, the NMD pathway has been investigated by studying the homologs of these genes. We present here the first unbiased genetic screen in Drosophila designed specifically to identify genes involved in NMD. By using a highly efficient genetic mosaic approach, we have screened ∼40% of the Drosophila genome and isolated more than 40 alleles of genes required for NMD. We focus on alleles we have obtained in two known NMD components: Upf2 and Smg6. Our analysis of multiple alleles of the core NMD component Upf2 reveals that the Upf2 requirement in NMD may be separate from its requirement for viability, indicating additional critical cellular roles for this protein. Our alleles of Smg6 are the first point mutations obtained in Drosophila, and we find that Smg6 has both endonucleolytic and nonendonucleolytic roles in NMD. Thus, our genetic screens have revealed that Drosophila NMD factors play distinct roles in target regulation, similar to what is found in mammals, but distinct from the relatively similar requirements for NMD genes observed in C. elegans and yeast.

The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila.

In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

The nonsense-mediated decay pathway maintains synapse architecture and synaptic vesicle cycle efficacy.

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol-3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant-negative or deleterious gain-of-function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain the efficacy of synaptic function.

Functions of the nonsense-mediated mRNA decay pathway in Drosophila development.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila "photoshop" mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3' UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.

Reactivation Assay to Identify Direct Targets of the Nonsense-Mediated mRNA Decay Pathway in Drosophila.

Transcriptome analysis provides a snapshot of cellular gene expression and is used to determine how cells and organisms respond to genetic or environmental changes. Identifying the transcripts whose expression levels are regulated directly by the manipulation being examined from those whose expression changes as a secondary cause from the primary changes requires additional analyses. Here we present a technique used to distinguish direct targets of the nonsense-mediated mRNA decay (NMD) pathway in Drosophila from secondary gene expression effects caused by loss of this pathway. This technique uses pulsed reexpression of an essential NMD gene in Drosophila lacking this NMD factor, followed by analysis of the transcriptome over time. In this way, RNAs with a rapid reduction in expression upon reactivation of NMD activity, corresponding to primary NMD targets, can be identified. This technique could potentially be modified to identify direct targets of other mRNA decay mechanisms in Drosophila or other organisms.

Multiple Nonsense-Mediated mRNA Processes Require Smg5 in Drosophila.

The nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway is a cellular quality control and post-transcriptional gene regulatory mechanism and is essential for viability in most multicellular organisms . A complex of proteins has been identified to be required for NMD function to occur; however, there is an incomplete understanding of the individual contributions of each of these factors to the NMD process. Central to the NMD process are three proteins, Upf1 (SMG-2), Upf2 (SMG-3), and Upf3 (SMG-4), which are found in all eukaryotes, with Upf1 and Upf2 being absolutely required for NMD in all organisms in which their functions have been examined. The other known NMD factors, Smg1, Smg5, Smg6, and Smg7, are more variable in their presence in different orders of organisms and are thought to have a more regulatory role. Here we present the first genetic analysis of the NMD factor Smg5 in Drosophila Surprisingly, we find that unlike the other analyzed Smg genes in this organism, Smg5 is essential for NMD activity. We found this is due in part to a requirement for Smg5 in both the activity of Smg6-dependent endonucleolytic cleavage, as well as an additional Smg6-independent mechanism. Redundancy between these degradation pathways explains why some Drosophila NMD genes are not required for all NMD-pathway activity. We also found that while the NMD component Smg1 has only a minimal role in Drosophila NMD during normal conditions, it becomes essential when NMD activity is compromised by partial loss of Smg5 function. Our findings suggest that not all NMD complex components are required for NMD function at all times, but instead are utilized in a context-dependent manner in vivo.

Degradation of Gadd45 mRNA by nonsense-mediated decay is essential for viability.

The nonsense-mediated mRNA decay (NMD) pathway functions to degrade both abnormal and wild-type mRNAs. NMD is essential for viability in most organisms, but the molecular basis for this requirement is unknown. Here we show that a single, conserved NMD target, the mRNA coding for the stress response factor growth arrest and DNA-damage inducible 45 (GADD45) can account for lethality in Drosophila lacking core NMD genes. Moreover, depletion of Gadd45 in mammalian cells rescues the cell survival defects associated with NMD knockdown. Our findings demonstrate that degradation of Gadd45 mRNA is the essential NMD function and, surprisingly, that the surveillance of abnormal mRNAs by this pathway is not necessarily required for viability.

In vivo determination of direct targets of the nonsense-mediated decay pathway in Drosophila.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a mRNA degradation pathway that regulates a significant portion of the transcriptome. The expression levels of numerous genes are known to be altered in NMD mutants, but it is not known which of these transcripts is a direct pathway target. Here, we present the first genome-wide analysis of direct NMD targeting in an intact animal. By using rapid reactivation of the NMD pathway in a Drosophila melanogaster NMD mutant and globally monitoring of changes in mRNA expression levels, we can distinguish between primary and secondary effects of NMD on gene expression. Using this procedure, we identified 168 candidate direct NMD targets in vivo. Remarkably, we found that 81% of direct target genes do not show increased expression levels in an NMD mutant, presumably due to feedback regulation. Because most previous studies have used up-regulation of mRNA expression as the only means to identify NMD-regulated transcripts, our results provide new directions for understanding the roles of the NMD pathway in endogenous gene regulation during animal development and physiology. For instance, we show clearly that direct target genes have longer 3' untranslated regions compared with nontargets, suggesting long 3' untranslated regions target mRNAs for NMD in vivo. In addition, we investigated the role of NMD in suppressing transcriptional noise and found that although the transposable element Copia is up-regulated in NMD mutants, this effect appears to be indirect.

Examination of Drosophila larval tracheal terminal cells by light microscopy.

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Drosophila Zpr1 (Zinc finger protein 1) is required downstream of both EGFR and FGFR signaling in tracheal subcellular lumen formation.

The cellular and molecular cues involved in creating branched tubular networks that transport liquids or gases throughout an organism are not well understood. To identify factors required in branching and lumen formation of Drosophila tracheal terminal cells, a model for branched tubular networks, we performed a forward genetic-mosaic screen to isolate mutations affecting these processes. From this screen, we have identified the first Drosophila mutation in the gene Zpr1 (Zinc finger protein 1) by the inability of Zpr1-mutant terminal cells to form functional, gas-filled lumens. We show that Zpr1 defective cells initiate lumen formation, but are blocked from completing the maturation required for gas filling. Zpr1 is an evolutionarily conserved protein first identified in mammalian cells as a factor that binds the intracellular domain of the unactivated epidermal growth factor receptor (EGFR). We show that down-regulation of EGFR in terminal cells phenocopies Zpr1 mutations and that Zpr1 is epistatic to ectopic lumen formation driven by EGFR overexpression. However, while Zpr1 mutants are fully penetrant, defects observed when reducing EGFR activity are only partially penetrant. These results suggest that a distinct pathway operating in parallel to the EGFR pathway contributes to lumen formation, and this pathway is also dependent on Zpr1. We provide evidence that this alternative pathway may involve fibroblast growth factor receptor (FGFR) signaling. We suggest a model in which Zpr1 mediates both EGFR and FGFR signal transduction cascades required for lumen formation in terminal cells. To our knowledge, this is the first genetic evidence placing Zpr1 downstream of EGFR signaling, and the first time Zpr1 has been implicated in FGFR signaling. Finally, we show that down-regulation of Smn, a protein known to interact with Zpr1 in mammalian cells, shows defects similar to Zpr1 mutants.

A novel function for the PAR complex in subcellular morphogenesis of tracheal terminal cells in Drosophila melanogaster.

The processes that generate cellular morphology are not well understood. To investigate this problem, we use Drosophila melanogaster tracheal terminal cells, which undergo two distinct morphogenetic processes: subcellular branching morphogenesis and subcellular apical lumen formation. Here we show these processes are regulated by components of the PAR-polarity complex. This complex, composed of the proteins Par-6, Bazooka (Par-3), aPKC, and Cdc42, is best known for roles in asymmetric cell division and apical/basal polarity. We find Par-6, Bazooka, and aPKC, as well as known interactions between them, are required for subcellular branch initiation, but not for branch outgrowth. By analysis of single and double mutants, and isolation of two novel alleles of Par-6, one of which specifically truncates the Par-6 PDZ domain, we conclude that dynamic interactions between apical PAR-complex members control the branching pattern of terminal cells. These data suggest that canonical apical PAR-complex activity is required for subcellular branching morphogenesis. In addition, we find the PAR proteins are downstream of the FGF pathway that controls terminal cell branching. In contrast, we find that while Par-6 and aPKC are both required for subcellular lumen formation, neither Bazooka nor a direct interaction between Par-6 and aPKC is needed for this process. Thus a novel, noncanonical role for the polarity proteins Par-6 and aPKC is used in formation of this subcellular apical compartment. Our results demonstrate that proteins from the PAR complex can be deployed independently within a single cell to control two different morphogenetic processes.

Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice.

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.

Branching morphogenesis of the Drosophila tracheal system.

Many organs including the mammalian lung and vascular system consist 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 sequential sprouting of primary, secondary, and terminal branches from an epithelial sac of approximately 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. Genetic 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 cellular 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.