uninflatable: Biological Overview | References
| Gene name - uninflatable
Cytological map position - 27D1-27D3
Keywords - Notch binding protein, essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation during asymmetric mitosis, asymmetric targeting of the Sara endosomes, PNS, tracheal inflation
Symbol - uif
FlyBase ID: FBgn0031879
Genetic map position - chr2L:6,972,828-7,010,359
Classification - Calcium-binding EGF-like domain - CUB domain - C-type lectin
Cellular location - endosomal transmembrane protein
Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate. Previous studies have shown that Notch/Delta molecules internalized in the mother cell traffic through Smad anchor for receptor activation (Sara) endosomes and are directed to the pIIa daughter. This study shows that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. The data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis (Loubery, 2014).
The Notch signaling pathway plays multiple roles in organisms ranging from flies and worms to mammals. A powerful model system to elucidate the cell biology of Notch signaling is the Drosophila sensory organs. Each sensory organ precursor (SOP) cell divides asymmetrically to produce a pIIa cell and a pIIb daughter cell, which perform directed Notch signaling: pIIb signals to pIIa. Four independent endocytic mechanisms control asymmetric signaling in the SOP. These include asymmetric endocytic events mediated by the E3 ubiquitin ligase Neuralized, recycling endosomes, and the endocytic adaptors α- and γ-adaptin together with Numb (Loubery, 2014).
During SOP cytokinesis, a fourth mechanism involves a population of endosomes marked by the adaptor protein Sara. Sara endosomes contain as cargo a pool of endocytosed Notch and Delta molecules. Notch and Delta reach the Sara endosome 20 min after their endocytosis in the SOP; this pool is dispatched into pIIa during cytokinesis. In contrast, the pools of Notch in endosomal populations upstream (Rab5 early endosomes) or downstream (Rab7 late endosomes) of Sara endosomes are segregated symmetrically. The specific pool of Notch in Sara endosomes is relevant for signaling: it is cleaved in a ligand- and gamma-secretase-dependent manner to release the transcriptionally active Notch intracellular domain (NICD) in pIIa (Loubery, 2014).
A key question is what machineries control the asymmetric targeting of these endosomes. Is the cargo (the ligand Delta or its receptor Notch) playing a role on the specific targeting of these endosomes? To unravel the machinery regulating the behavior of Sara endosomes during SOP mitosis, candidate factors from previously reported proteomics approaches or genetic screens were tested for Notch signaling. Thus, Uninflatable was identified as a factor involved in the asymmetric dynamics of Sara endosomes (Loubery, 2014).
MARCM homozygous mutant clones were generated for a null allele of Uninflatable (Uif2B7) an the trafficking of Delta, Notch, and the Notch effector Sanpodo through Sara endosomes was monitored. To look at the motility of the endogenous population of Sara endosomes, the cohort of internalized Delta molecules 20 min after its endocytosis was followed in the SOP by means of a pulse-chase antibody uptake assay. Delta, Notch, and Sanpodo traffic normally through Sara endosomes in the absence of Uif, and these endosomes are targeted to the cleavage plane (the central spindle) in cytokinesis (Loubery, 2014).
In Uif mutants or RNAi knockdown conditions, iDl20'/Sara endosomes fail to be asymmetrically dispatched to pIIa after their targeting to the central spindle. These results indicate that Uif is not required to bring Notch to the Sara endosomes or to target the endosomes to the central spindle. However, once in the spindle, Uif is essential for the specific dispatch of Sara endosomes from the spindle into the pIIa cell (Loubery, 2014).
This function of Uninflatable is specific to the asymmetric segregation of Sara endosomes. To gain mechanistic insights into the mechanism of action of Uif, this study has analyzed the density of microtubules in the central spindle and has shown that Uninflatable does not regulate the organization of the microtubular cytoskeleton. In contrast, it was found that Uif controls the residence time of Sara endosomes on the central spindle: in control SOPs, Sara endosomes depart from the central spindle with a decay time of 103 ± 21 s, whereas upon Uif downregulation this decay time goes up to 175 ± 42 s. These data indicate that Uif is not involved in the organization of the spindle, but rather in the motility properties of the endosomes, particularly their last step of departing from the central spindle and end up in pIIa (Loubery, 2014).
Consistent with the role of Uif in the asymmetric targeting of Sara endosomes, Uif contributes to Notch-dependent cell fate assignation in the SOP lineage. To address this, the composition of SOP lineages was examined in homozygous Uif2B7 MARCM clones or upon Uif RNAi. In wild-type animals, the SOP lineage consists of four different cells: two external cells (the shaft and the socket) originating from pIIa and two internal cells (the sheath and the neuron) from pIIb, which can be identified by immunostaining. In Uif mutant clones, instead of a sheath and a neuron per SOP lineage, two sheath cells can be frequently observed in the notum, indicating a symmetric division in the pIIb lineage. Similarly, upon Uif downregulation in the postorbital SOPs, duplications of sockets were observed, which is diagnostic of symmetric divisions in the pIIa lineage. These data uncover a role for Uninflatable in Notch-dependent asymmetric cell fate assignation that is mediated by the asymmetric dispatch of the Sara endosomes (Loubery, 2014).
The Uif phenotype during asymmetric endosomal targeting and cell fate assignation prompted us to look whether Uif is a cargo of Sara endosomes. To detect the endogenous protein, anti-Uif antibodies were generated. To look at Uif trafficking in vivo, transgenic flies were generated expressing a Uif-GFP protein, which can provide activity to rescue the lethality of a Uif lethal mutation at least partly (Loubery, 2014).
Uif-GFP is strongly colocalized with both Sara-GFP and iDelta20'. Since a cargo of Sara endosomes is Notch itself (73% ± 2.7% of the vesicular population of Notch molecules is in Sara endosomes), the presence of Notch cargo was examined in Uif vesicles: 44% ± 4.7% of Uif-positive vesicular structures contain Notch. Therefore, a population of Uninflatable and Notch traffics through Sara endosomes during SOP asymmetric mitosis (Loubery, 2014).
The fact that Uninflatable controls the asymmetric dispatch of the Sara endosomes, which contain internalized Notch and Uninflatable, prompted a look at a possible molecular interaction between Uninflatable and Notch. Uif- and Notch-expressing plasmids were cotransfected in S2 cells and immunoprecipitation experiments were performed by using anti-Uif-coupled beads, followed by immunoblotting with a clean anti-Notch antibody that was purified from a hybridoma cell line (DSHB #C17.9C6). Uif was shown to immunoprecipitate Notch. This coimmunoprecipitation can be reproduced from lysates of S2 cells expressing Notch and Uif tagged with the PC peptide tag and anti-PC-coupled beads; as a control, other transmembrane proteins such as Tkv-GFP are not coimmunoprecipitated with Uif-PC. Together, these results indicate a specific molecular interaction between Notch and Uif (Loubery, 2014).
Uninflatable is a transmembrane protein that, like Notch, contains an array of epidermal growth factor (EGF) repeats. It has been shown that Notch is engaged in protein-protein interactions through its EGF repeats with other factors containing EGF repeats. These include its ligand Delta, but also a number of noncanonical Notch ligands, secreted or membrane proteins lacking the DSL domain characteristic of canonical Notch ligands (Dlk-1, Dlk-2, DNER, Trombospondin, LRP1, EGFL7, and Weary). Consistently, it has recently been reported that a synergistic genetic interaction between Uif and Notch depends on Notch EGF repeats. Therefore, studies were performed to discover which EGF repeats of Uif could be involved in the molecular interaction with Notch. A coimmunoprecipitation experiment was performed in S2 cells coexpressing Notch and an N-terminal, truncated form of Uif tagged with PC (UifΔCter-PC) that lacks the four EGF domains flanking the transmembrane domain but still contains the other 17 EGF repeats and other extracellular domains. While full-length Uif-PC coimmunoprecipitates Notch, UifΔCter-PC does not. This indicates that the interaction between Uif and Notch may be mediated by the four EGF domains of Uif flanking its transmembrane domain (Loubery, 2014).
Although Uif binds and colocalizes with Notch, it does not play a role in core Notch signaling: embryos deprived of maternal and zygotic Uif in germline clones do not show a Notch signaling phenotype, whereas they display loss of inflation of the trachea as previously reported. Consistently, loss of Uif in wing mosaics does not cause a defect of Notch-dependent expression of Wingless at the wing margin. This indicates that Uninflatable interaction with Notch is not essential during core Notch signaling, but rather during the asymmetric dispatch of Notch-containing Sara endosomes during asymmetric cell division. This prompted the possibility that Notch itself is required for the asymmetric motility of the endosomes (Loubery, 2014).
To study whether Notch plays a role during the asymmetric dispatch of Sara endosomes, the trafficking was studied of a Notch-GFP fusion expressed at endogenous levels. The idea was to confirm previous observations using a Notch antibody uptake assay to follow Notch expressed at endogenous levels. To achieve this, a reporter transgenic fly strain was set up in which Notch-GFP fusion is driven by the Notch endogenous promoter and is expressed at endogenous levels. In this fusion, GFP is inserted in the middle of the Notch-intra domain. Since in protein fusions GFP is frequently cleaved out, whether the fusion protein is intact was examined. This would be particularly important in this case, since a cleavage event would lead to a truncated Notch-intra peptide (Loubery, 2014).
In these transgenic Notch-GFP flies, GFP is very efficiently cleaved out (74% of total GFP is cleaved, leading to truncated Notch-intra peptides that can only partially support Notch function and thereby cause a highly penetrant mutant phenotype. This precludes the usage of this reagent as a bona fide marker for Notch. In particular, the cytosolic GFP signal cannot be used as a readout of signaling as previously reported: a nuclear accumulation of the GFP signal in these flies does not solely reflect the accumulation of Notch-intra-GFP, but rather the overall accumulation of different GFP-containing fragments (Loubery, 2014).
Whether, in these conditions, the pool of membrane associated GFP-Notch traffics through Sara endosomes and is asymmetrically dispatched to the pIIa cell was studied. Only 11% ± 1.3% of the total GFP signal in these flies is membrane associated (plasma membrane and intracellular vesicular structures). The rest, representing the vast majority (89%), corresponds to cytosolic and nuclear cleaved GFP (Loubery, 2014).
In Notch-GFP flies, 3.1% of the total GFP signal is associated with intracellular vesicular structures. These correspond to various intracellular vesicular compartments, including Notch in the secretory pathway, as well as in early endosomes, Sara endosomes, recycling endosomes, and late endosomes. To measure the size of the specific pool of Notch in Sara endosomes, a Notch antibody internalization assay was performed, and internalized Notch was chased 20 min after its endocytosis (iNotch20'). As previously established, 73% ± 2.7% of Notch-GFP vesicles are positive for iNotch20'. Of this iNotch20'-positive pool, 79% would be targeted to pIIa . This is consistent with only 65% ± 3.1% of the total pool of Notch-GFP being dispatched to pIIa (Loubery, 2014).
Whether Notch itself plays a role on the asymmetric targeting of Sara endosomes was addressed. Notch was depleated in the SOP by expressing a previously validated Notch dsRNA, and the behavior of Sara endosomes was examined. Upon Notch knockdown in the SOP, iDl20'/Sara endosomes are still targeted to the central spindle, but the subsequent directed dispatch to pIIa is defective. This indicates that Notch itself contributes to the endosomal recruitment of the machinery that endows the Sara endosomes with their asymmetric behavior (Loubery, 2014).
It has been shown that the targeting of Notch to Sara endosomes does not depend on Uninflatable; it was then determined whether the recruitment of Uninflatable on Sara endosomes depended on Notch. Interestingly, it was found that, conversely, the targeting of Uif to Sara endosomes is not controlled by Notch. This implies that these two molecules use different machineries to get to the endosome, where they can interact and are both required for the asymmetric motility of the endosome (Loubery, 2014).
Since the Notch receptor itself is required for the asymmetric targeting of Sara endosomes, it was asked whether Notch signaling plays a role in the process. Notch signaling was blocked by inactivating the ligand Delta through overexpression of Tom in the SOP cell; Tom overexpression leads to inactivation of the Ubiquitin ligase Neuralized and thereby blocks endocytosis-dependent activation of Delta. In the absence of Notch signaling, targeting of Sara endosomes to the central spindle and their asymmetric dispatch to the pIIa cell remains intact. This indicates that although the Notch receptor is essential for the asymmetric targeting of Sara endosomes, Notch signaling is not (Loubery, 2014).
This report has started to unravel the machinery that mediates asymmetric endosome motility during asymmetric cell division. Both Notch and Uninflatable were shown to play a key role in the last step of the asymmetric motility of endosomes: the final, specific stride of the Sara endosomes from the central spindle into the anterior pIIa cell. This is based on the following four key sets of observations (Loubery, 2014).
First, it was confirmed that a functional Notch-GFP fusion expressed at endogenous level does traffic through Sara endosomes, which are indeed dispatched asymmetrically during SOP mitosis. Second, Notch binds Uninflatable, and both colocalize in Sara endosomes. Third, neither Notch nor Uninflatable is essential for the targeting of Notch/Delta/Uif to the Sara endosomes or the targeting of those endosomes to the central spindle, but they are essential for the final dispatch from the central spindle into the pIIa cell. Although Notch is necessary for this process, Notch signaling is not. Fourth, Uninflatable is not an integral component of the Notch signaling pathway, but it plays a role during asymmetric Notch signaling in the SOP, and therefore mutant Uif conditions lead to a lineage identity phenotype. It remains to be elucidated what machineries downstream of Notch/Uninflatable implement the control of the final step toward pIIa and what is asymmetrical in the cytoskeleton so that this final step occurs toward pIIa and not pIIb (Loubery, 2014).
Notch signaling is a highly conserved pathway in multi-cellular organisms ranging from flies to humans. It controls a variety of developmental processes by stimulating the expression of its target genes in a highly specific manner both spatially and temporally. The diversity, specificity and sensitivity of the Notch signaling output are regulated at distinct levels, particularly at the level of ligand-receptor interactions. This paper reports that the Drosophila gene uninflatable (uif), which encodes a large transmembrane protein with eighteen EGF-like repeats in its extracellular domain, can antagonize the canonical Notch signaling pathway. Overexpression of Uif or ectopic expression of a neomorphic form of Uif, Uif*, causes Notch signaling defects in both the wing and the sensory organ precursors. Further experiments suggest that ectopic expression of Uif* inhibits Notch signaling in cis and acts at a step that is dependent on the extracellular domain of Notch. these results suggest that Uif can alter the accessibility of the Notch extracellular domain to its ligands during Notch activation. This study shows that Uif can modulate Notch activity, illustrating the importance of a delicate regulation of this signaling pathway for normal patterning (Xie, 2012).
The canonical Notch signaling pathway is one of a limited group of pathway modules that transduce signals from outside the cell to alter gene expression inside the nucleus. These pathways together orchestrate the developmental processes that can be dauntingly complex. Yet it is the same modules that are used repeatedly, not only in different organisms, but also in vastly different processes within an organism. Thus, how these pathway modules are activated in a specific manner, with regard to not only space and time but also the quantity of their signaling output, represents a fundamental question in developmental biology. This study describes a newly characterized protein, Uif, which can antagonize the canonical Notch signaling pathway in a neomorphic manner. These findings underscore the importance of the precise tuning of Notch activity in normal patterning (Xie, 2012).
EGF-like repeats are a common feature of Notch receptors, ligands and co-ligands. While Uif was originally characterized for its role in tracheal development, its EGF-like repeats suggest a possible role in Notch signaling. The current results are consistent with a model where ectopically expressed Uif may modulate the accessibility of the extracellular domain of Notch to its ligands during activation. It is possible that the EGF-like repeats of Uif directly interact with the extracellular domain of Notch to exert its inhibitory effect in a manner similar to the cis inhibition by Notch ligands themselves. The finding that Uif* acts on Notch through a cis inhibitory mechanism is supportive of this possibility. In the current experiments, Uif* is more effective than wt Uif in antagonizing Notch, and this difference may be attributed to the difference in their expression levels. These results suggest that ectopically expressed Uif* and wt Uif have a similar neomorphic function in regulating Notch signaling (Xie, 2012).
A proposed neomorphic function of Uif* and Uif in Notch signaling is consistent with the results of loss of function analysis of uif. Knockdown (assayed for adult wing phenotypes and Notch target gene expression using independent RNAi lines) or knockout (assayed for Notch target gene expression in somatic mutant clones) of uif revealed neither Notch loss of function nor gain of function phenotypes. However, it remains formally possible that the endogenous uif gene has a native role in regulating Notch signaling in tissues or cells (other than those examined in this study) at a time during Drosophila development. Further studies are required to investigate this possibility (Xie, 2012).
The biological activities of Uif are not restricted to regulating Notch signaling. The fact that Uif was originally characterized for its role in tracheal inflation underscores the complexity of its biological activities. In addition to the EGF-like repeats, Uif also contains several other domains that may have important biological functions. These domains include a C-type lectin-like (CLECT) domain, three CUB domains, eight complement control protein (CCP) domains, two coagulation factor 5/8 C-terminal (FA58C) domains and three hyaline repeat (HYR) domains. Both CLECT and FA58C domains are putative carbohydrate binding domains known to play important roles in many diverse processes. The CUB domain is an evolutionary conserved protein domain found almost exclusively in extracellular and plasma membrane-associated proteins. HYR is an immunoglobulin fold domain likely involved in cell adhesion. The CCP domains, also known as the Sushi domains or Short Consensus Repeats (SCR), exist in a wide variety of complement and adhesion proteins. These domains suggest that Uif may also play a role in cell adhesion. Indeed, in a recent genetic modifier screen, uif was identified as a regulator (Mod29) of the Drosophila Dystroglycan-Dystrophin Complex, a specialized cell adhesion complex (Kucherenko, 2008). Mod29/Uif was suggested to play roles in multiple developmental processes, including wing vein formation, muscle and photoreceptor axon development, and oogenesis (Kucherenko, 2008). Although it remains to be investigated whether Uif, a large regulator with multiple conserved protein domains, may functionally connect distinct cellular processes, unpublished data offer some speculative insights. In particular, the blistering wing phenotype caused by knockdown of Dl or Ser can be fully rescued by depletion of uif, suggesting that Uif may functionally extend the role of Notch ligands to cell adhesion. Uif is an N-glycosylated protein, a modification shared by several proteins known to play a role in the formation of large protein complexes. Understanding the full spectrum of the biological functions of Uif during development and, importantly, its potential role in harmonizing different cellular processes, represents future challenges (Xie, 2012).
The tracheal system of Drosophila has proven to be an excellent model system for studying the development of branched tubular organs. Mechanisms regulating the patterning and initial maturation of the tracheal system have been largely worked out, yet important questions remain regarding how the mature tubes inflate with air at the end of embryogenesis, and how the tracheal system grows in response to the oxygen needs of a developing larva that increases nearly 1000-fold in volume over a four day period. This study describes the cloning and characterization of uninflatable (uif), a gene that encodes a large transmembrane protein containing carbohydrate binding and cell signaling motifs in its extracellular domain. Uif is highly conserved in insect species, but does not appear to have a true ortholog in vertebrate species. uif is expressed zygotically beginning in stage 5 embryos, and Uif protein localizes to the apical plasma membrane in all ectodermally derived epithelia, most notably in the tracheal system. uif mutant animals show defects in tracheal inflation at the end of embryogenesis, and die primarily as larvae. Tracheal tubes in mutant larvae are often crushed or twisted, although tracheal patterning and maturation appear normal during embryogenesis. uif mutant larvae also show defects in tracheal growth and molting of their tracheal cuticle (Zhang, 2009).
This study has described the initial characterization of a novel gene, uninflatable, which encodes a single-pass type I transmembrane protein with several different carbohydrate binding motifs and epidermal growth factor repeats in its extracellular domain. This gene is highly conserved in insect species, but does not appear to have a true ortholog in mammals or other vertebrate organisms. Uninflatable is expressed on the apical surface of ectodermally-derived epithelial cells including the epidermis, trachea, salivary gland and fore- and hindgut, although it is most highly expressed in tracheal epithelia starting as the tracheal placode invaginates and persisting throughout its development. Three mutant alleles of uif were isolated, and the predominant defects associated with loss of uif were found to be defective air inflation at the end of embryogenesis, and tracheal growth and tracheal molting defects in larvae (Zhang, 2009).
The most obvious defects observed in uif mutant late embryos and newly hatched first instar larvae were incomplete inflation of the trachea coupled with tracheal tubes that often appeared pinched or stretched. Live imaging of individual uif mutant embryos and larvae revealed that these tracheal inflation defects result from the inability to completely inflate the trachea at the end of embryogenesis rather than a defect in the maintenance of an inflated tracheal system after hatching. Interestingly, tracheae in uif mutant embryos are normally patterned and appear to mature in a manner indistinguishable to tracheae in wild type animals. Thus this tracheal inflation defect likely results from a requirement for uif function late during embryogenesis (Zhang, 2009).
Several genes have been characterized whose defects include incomplete tracheal inflation. Included in these genes are those that function at the end of embryogenesis to clear the trachea of solid luminal material. This process requires clathrin-mediated endocytosis and includes the proteins clathrin heavy chain, the GTPase dynamin (encoded by shibire), and a clathrin binding transmembrane protein encoded by wurst. Notably, mutations in all of these genes also result in elongated tracheal tubes suggesting a defect in tracheal tube size control. Gas filling defects were also observed for mutations in serp and verm, two putative matrix chitin deacytlases that function to regulate tracheal tube length. Tracheal maturation appears normal in uif mutant embryos, including the secretion of chitin into the tracheal lumen and the subsequent uptake of luminal solids near the end of embryogenesis. In addition, uif mutant tracheae do not show diameter or length defects during embryogenesis, suggesting that the luminal chitin cylinder forms normally and that Serp and Verm are correctly functioning during the maturation process. Taken together these results suggest that the air filling defects observed in uif mutant embryos occurs through a mechanism independent of that linked to endocytosis of solid luminal material (Zhang, 2009).
A second class of proteins that likely function in proper air inflation is the epithelial sodium channels (ENaCs). In mammals, ENaC proteins are required to help remove liquid from embryonic lung prior to birth. There are 16 ENaC genes (also referred to as pickpocket or ppk genes) in the Drosophila genome, of which 9 are expressed in the embryonic tracheal system. It is thought that the influx of sodium through these channels drives water from the lumen into the epithelial cells, and that degassing of this liquid inflates the trachea. Although there are no loss of function mutations in ENaCs that result in air filling defects in Drosophila, RNA interference mediated knockdown of ppk4 and ppk11 results in partially liquid-filled tracheae in larvae. In addition, inhibiting these channels using amiloride also results in fluid filled tracheae in larvae. Although both of these examples affected air filling after a molt, it seems likely that ENaC proteins may also contribute to air filling at the end of embryogenesis. At this point it is not possible to exclude the possibility that uif may regulate the expression or activity of ENaC encoded proteins in tracheal epithelia (Zhang, 2009).
A third possibility, and one that is favored, is that uif is playing primarily a structural role in embryonic tracheal maturation. Two pieces of evidence support this notion. First, in early stage 17 mutant embryos the tracheae are of normal length and diameter and have a stereotypic appearance that is indistinguishable from that of wild type animals, whereas in newly hatched mutant larvae the tubes are often crushed or twisted. Second, while dissecting tracheae from wild type and uif mutant third instar larvae it was clear that the mutant tracheae were more brittle. Wild type tracheae have an elastic property that makes them difficult to break, whereas we had to be very careful dissecting uif tracheae in order to get a section that included more than one metameric unit. Together these observations suggested a model in which the mechanical properties of uif mutant tracheae are compromised, thereby allowing the tracheal cuticle to fail when the embryo initiates violent muscular contractions prior to hatching. As noted in the live imaging, tracheal inflation initiates well after the embryo begins these dramatic muscular contractions. It is predicted that a combination of crushed tubes and small cracks in the tracheal cuticle prevent complete inflation (Zhang, 2009).
uif mutant larvae that survive to second or third instar show striking defects in tracheal growth and tracheal molting. The tracheae in these uif mutant larvae have short dorsal trunks that are well out of proportion to the body length of the animal. It is difficult to accurately assess the diameter of the dorsal trunk in these animals, however, because the mutant animals fail to completely shed their tracheal cuticle, and often only the first instar lumen is inflated. This tracheal molting defect was nearly completely penetrant in all third instar uif2B7 and uif1A15 mutant larvae examined, but the severity seemed to vary along the anterior-posterior axis, with some middle and anterior sections showing no molting defects, whereas the sections near the posterior spiracles showed at least two and sometimes three tracheal cuticles. A previous study carried out morphometric analyses of larval tracheal growth and observed that tracheae lengthen in a continuous fashion, whereas the diameter increases in a stepwise manner coincident with the molt, suggesting that tube length is controlled independently from tube diameter. The current results indicate that uif plays a role in regulating the growth of the tracheae along their length (Zhang, 2009).
How might uif regulate larval tracheal tube length? One possibility is that Uif might regulate the interface between the tracheal epithelium and the cuticle, possibly by providing lubrication through its hyalin domains. Loss of uif would therefore result in a situation where the epidermis is too tightly bound to the overlying cuticle to allow for growth along the long axis of the tracheae. This type of mechanism could also account for the tracheal molting defects and thereby couple these phenotypes. An earlier study identified mutations in the Matrix Metalloproteinase encoded by Mmp1; similar defects were found in tracheal elongation. It was speculated that the Mmp1 tracheal defects might be caused by the inability of the tracheal epithelial cells to loosen their attachment to the cuticle. Since uif2B7 mutant larvae have no full length Uif protein, it is unlikely that Uif is a primary target of MMP1 in tracheal cells. In addition, this study observed no genetic interaction between uif and Mmp1 by second-site noncomplementation (Zhang, 2009).
An alternative hypothesis is that Uif might serve as a receptor or co-receptor for a systemic signal that couples tracheal growth with larval growth. The extracellular domain of Uif contains multiple EGF domains and a laminin G domain, and the cytoplasmic domain is remarkably conserved in all insect species. A combination of structure/function analysis and genetic and biochemical approaches to identify Uif interacting proteins should help to shed light on this functions of uif. Understanding how Uif couples tracheal growth to the growth of the larva may serve as an important paradigm for similar couplings of organ growth to organismal growth in other species (Zhang, 2009).
Finally, hypoxia may account for all the other larval phenotypes associated with loss of uif, including early larval lethality, slow growth, developmental arrest and failure to pupariate. Most uif mutant larvae die as first instars, and those that die early almost invariably have the most severe tracheal inflation defects. uif mutant larvae that survive to second or third instar grow much slower than their heterozygous siblings. btl>uifRNAi larvae that have only lost uif function in their tracheae show an identical slow growth phenotype. This study consistently observed uif mutant larvae wandering away from food, suggesting that they were experiencing hypoxia. Not surprisingly these larvae accumulate less fat and have a transparent appearance. Examination of the tracheae in these mutant animals revealed that the inflated portion of the trachea was often just through the first instar tracheal lumen, and therefore these animals were likely oxygen starved as well. Consistent with this notion, additional tracheal branching was observed in these mutant animals suggesting that the hypoxia induced factor pathway had been engaged. Thus, hypoxia could explain the growth defects observed in uif mutant larvae. These growth defects may have then resulted in the observed developmental delays. For example, mutant third instar larvae may not have reached a critical weight threshold needed for pupariation, and thus would not have been able to pupariate even if they experienced the metamorphic pulse of ecdysone. Interestingly, many uif mutant animals even failed to advance to third instar, but this did not reflect a defect in epidermal molting or ecdysis, as no evidence was found for an extra set of head skeleton or epidermal cuticle. Rather the animals just arrested as first or second instars. It is possible that hypoxia-induced growth defects contributed to this phenotype as well, since larvae have to attain a critical size to be competent for molting, just as they do for pupariation (Zhang, 2009).
Search PubMed for articles about Drosophila Uninflatable
Kucherenko, M. M., Pantoja, M., Yatsenko, A. S., Shcherbata, H. R., Fischer, K. A., Maksymiv, D. V., Chernyk, Y. I. and Ruohola-Baker, H. (2008). Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex. PLoS One 3: e2418. PubMed ID: 18545683
Loubery, S., Seum, C., Moraleda, A., Daeden, A., Furthauer, M. and Gonzalez-Gaitan, M. (2014). Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division. Curr Biol 24: 2142-2148. PubMed ID: 25155514
Xie, G., Zhang, H., Du, G., Huang, Q., Liang, X., Ma, J. and Jiao, R. (2012). Uif, a large transmembrane protein with EGF-like repeats, can antagonize Notch signaling in Drosophila. PLoS One 7: e36362. PubMed ID: 22558447
Zhang, L. and Ward, R. E. t. (2009). uninflatable encodes a novel ectodermal apical surface protein required for tracheal inflation in Drosophila. Dev Biol 336: 201-212. PubMed ID: 19818339
date revised: 28 December 2015
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