Netrin-A and Netrin-B

Netrins guide Drosophila commissural axons at short range

Netrins are secreted axon guidance molecules required for commissure formation in a wide range of animal species, including C. elegans, Drosophila melanogaster and mice. They are generally thought to function as chemoattractants, acting at a distance to direct commissural axon growth toward the midline of the central nervous system. This study shows, however, that D. melanogaster commissural axons still orient normally and reach the midline even in the complete absence of netrins, though some of them fail to cross the midline. Tethering endogenous netrin to the membrane selectively disrupts its long-range but not short-range activity, yet still allows normal commissure formation. It is therefore proposed that netrins act in commissural axon guidance as short-range cues that promote midline crossing, not as long-range chemoattractants (Brankatschk, 2006).

Netrins are secreted axon guidance molecules best known for their phylogenetically conserved role in the formation of the commissural axon pathways that extend across the midline of the central nervous system. Netrins are expressed by midline cells, and have generally been thought to guide commissural axons toward the midline by long-range chemoattraction. Data presented here, however, led to the proposal of a very different function for netrins in the ventral nerve cord of the Drosophila embryo. Specifically, rather than being long-range cues to attract commissural axons toward the midline, it is believed that netrins are short-range cues that help some of these axons to extend across it. This conclusion rests on data from two sets of experiments: (1) single-cell labeling experiments indicate that axons still orient correctly and grow toward the midline in the complete absence of netrin; (2) tethering netrin to the membrane of the cells that produce it selectively disrupts its long-range but not its short-range activity, yet leaves commissures intact (Brankatschk, 2006).

DiI labeling was used to examine axonal trajectories in wild-type and netrin-deficient embryos. This method allowed a systematic survey a large neuronal population at single-cell resolution. Analysis focused on lateral neurons, in the belief that they should be the most sensitive to the loss of any long-range chemoattractant from the midline. However, the only apparent defect among these neurons is a failure of a small fraction of commissural axons to cross the midline in the posterior commissure, although they evidently approach it normally. Nevertheless, the frequency of such errors, examined at single-cell resolution, is too low to explain the highly penetrant disruption of the posterior commissure when all axons are visualized simultaneously. This suggests that, on the whole, lateral neurons are relatively unaffected by the loss of netrin function, and that the commissural defects in netrin mutants are more likely due to misrouting of medial rather than lateral neurons. DiI labeling is technically much more challenging for medial neurons, and this was not tested systematically. However, it is noted that, of the neurons examined using a genetic marker (eg-GAL4), the medial neurons are indeed much more severely affected than the lateral neurons (55% errors by medial EW neurons compared to 8% errors by lateral EG neurons). This pattern is not what one would predict to observe upon the loss of a long-range cue for midline attraction. A possible explanation for the greater sensitivity of medial axons to the loss of netrins is that, if netrins are indeed short-range cues for midline crossing, then lateral but not medial neurons would need some other mechanism to direct their initial axon growth toward the midline. For these lateral neurons, this mechanism alone may often suffice for midline crossing, even in the absence of the short-range signal provided by netrins (Brankatschk, 2006).

These experiments in which NetB was tethered to the membrane were inspired by similar experiments previously used to discriminate between the short- and long-range activities of the morphogens Wingless and Hedgehog. Whereas these previous experiments involved the use of transgenes to ectopically express membrane-tethered proteins, gene targeting was used to attach the membrane anchor directly to the endogenous NetB protein. In the cases of Wingless and Hedgehog, membrane-tethering abolished their long-range activities, but still allowed them to signal to adjacent cells (Brankatschk, 2006).

Similarly, membrane tethering abolishes the ability of NetB to repel Unc5-expressing axons at a distance, but still allows NetB to repel axons at close range. Remarkably, commissures still form almost normally in these embryos, despite the membrane tethering of NetB and the complete absence of NetA. Thus, although NetB can guide Unc5-expressing axons at long range, it evidently guides commissural axons only at short range. Taken together, the DiI labeling and membrane-tethering experiments provide strong evidence that in Drosophila, netrins are local cues for commissural axons, not long-range chemoattractants (Brankatschk, 2006).

If this interpretation is correct, then it raises the obvious question of what, then, does direct lateral commissural axons toward the midline. Three possibilities can be envisioned. (1) Midline cells might provide some other chemoattractant. Sonic hedgehog seems to have just such a role in vertebrates, but Drosophila Hedgehog is not expressed by midline cells and so is unlikely to have an analogous function in commissure formation. (2) Axons might grow toward the midline in response to a lateral repellent, rather than a midline attractant. In vertebrates, members of the bone morphogenic protein (BMP) family act as dorsal repellents to direct commissural axons ventrally, and Slit/SLT-1 has a similar function in C. elegans. Yet, again, it seems unlikely that either a BMP protein or Slit acts as a lateral repellent for commissural axons in D. melanogaster. The existence of additional attractants or repellents for commissural axons in D. melanogaster thus remains pure conjecture. (3) The initial growth of lateral axons may instead be controlled locally, by polarity cues that act at the cell body. These polarity signals might act directly to influence the site and orientation of the initial axon outgrowth, or indirectly by determining the overall polarity of the neuron or its precursor. If commissural axons were oriented medially by such local polarity cues, then they might reach the midline by purely intrinsic mechanisms that promote continued growth in the same direction. This latter model is particularly appealing since it obviates the need for any long-range guidance to occur at all (Brankatschk, 2006).

Finally, just as the experiments were inspired by studies on long-range signaling by morphogens, it is hoped that the experiments will in turn inspire further examination of the long-range signaling activities of guidance factors in other contexts. The observations caution against the simple inference that a guidance factor that can move away from its source and influence axon growth at a distance, necessarily does so in all cases. Experiments aimed at discriminating the short- and long-range activities of axon guidance factors, and understanding how any long-range action is exerted, will surely be as informative for chemoattractants and chemorepellents as they have been, and continue to be, for morphogens (Brankatschk, 2006).

Intra-axonal patterning: intrinsic compartmentalization of the axonal membrane in Drosophila neurons

In the developing nervous system, distribution of membrane molecules, particularly axon guidance receptors, is often restricted to specific segments of axons. Such localization of membrane molecules can be important for the formation and function of neural networks; however, how this patterning within axons is achieved remains elusive. This study shows that Drosophila neurons in culture establish intra-axonal patterns in a cell-autonomous manner; several membrane molecules localize to either proximal or distal axon segments without cell-cell contacts. This distinct patterning of membrane proteins is not explained by a simple temporal control of expression, and likely involves spatially controlled vesicular targeting or retrieval. Mobility of transmembrane molecules is restricted at the boundary of intra-axonal segments, indicating that the axonal membrane is compartmentalized by a barrier mechanism. It is proposed that this intra-axonal compartmentalization is an intrinsic property of Drosophila neurons that provides a basis for the structural and functional development of the nervous system (Katsuki, 2009).

This study describes a patterning phenomenon that takes place within single axonal processes as a cell-intrinsic event. This patterning involves compartmentalization of the axonal membrane with a diffusion barrier located at a medial point of the axon. The data suggest that this patterning ability is a fundamental property of Drosophila neurons, because the compartment-specific localization of GFP-tagged receptors can be observed in the majority (>90%) of neurons. In the CNS of Drosophila, more than 90% of neurons project their axons to the contralateral side of the nervous system, and the width of the commissural segment or precrossing segment of those neurons is 20-40 μm, which parallels the length of the proximal compartment observed in vitro. This raises the possibility that the intrinsic patterning ability of neurons may serve as the basis of generating the intra-axonal localization of guidance molecules in vivo (Katsuki, 2009).

In addition to these intrinsic abilities of neurons, the results suggest that extrinsic factors may also contribute to the intra-axonal patterning, because not all ROBO receptors examined in this study recapitulated the localization patterns observed in vivo. All three members of ROBO family receptors are localized to distal axon in vivo. Whereas ROBO2 and ROBO3 retained the ability to localize distally when isolated in culture, ROBO was uniformly distributed along axons under such conditions. Localization of ROBO may require extrinsic signals that are absent in the culture system. One of the candidate extrinsic factors are the midline cells, which lie on an axonal region where ROBO expression is downregulated in vivo. It is also possible that the location of the compartment boundary determined by the intrinsic mechanisms is refined by extrinsic signals. It would be interesting to test whether contact with midline cells in culture can induce distal localization of ROBO, or alter the position of the boundary (Katsuki, 2009).

It has been commonly suggested that axon guidance receptors are targeted to the growth cone, and intra-axonal localization patterns of guidance receptors reflect temporal profiles of receptor expression at the growth cone during axonal extension. This study demonstrates that intra-axonal localization patterns that are evident in the culture condition can form regardless of the timing of receptor expression. Although this result does not rule out the involvement of temporal control of expression during axon navigation in vivo, it suggests that critical mechanisms for the intra-axonal localization of receptors described in this study are compartment-specific trafficking pathways. One such trafficking mechanism could involve local translation or targeted membrane transport, which can specifically deposit membrane proteins to either the proximal or distal membrane compartment. It is also possible that membrane proteins are selectively retrieved from one compartment through endocytic pathways (Katsuki, 2009).

A time course experiment in shi^ts1 mutant backgrounds suggests that Derailed (DRL) is preferentially targeted to the proximal compartment. It was also shown that the correct intra-axonal localization of DRL requires Dynamin-dependent endocytosis; however, at present it cannot be distinguished whether or not the endocytosis of DRL is compartment specific. Because fluorescence recovery after photobleaching (FRAP) experiments on CD8-GFP suggest that the barrier between the proximal and distal axon compartments does not completely block the movement of membrane proteins between the compartments, it is possible that the Dynamin-dependent endocytosis is required to remove DRL that leaks into the distal compartment, serving to maintain the pattern generated by targeting. Alternatively, endocytosis itself may be compartment specific, contributing to the establishment of the pattern (Katsuki, 2009).

In contrast to DRL, ROBO3 does not appear to require shibire function for its localization, demonstrating the presence of differential trafficking mechanisms for DRL and ROBO3. Due to this shi-independence of ROBO3, it is not possible to conclusively demonstrate the presence of preferential targeting of ROBO3 by performing a time course experiment. Even if there is preferential targeting, it is likely that ROBO3 also needs to be removed from the incorrect compartment, because ROBO3 shows a level of lateral mobility on the axon similar to that of DRL. Since ROBO3 localization is largely independent of Dynamin function, such a retrieval pathway must be based on Dynamin-independent mechanisms. While the complementary localization patterns of DRL and ROBO3 suggests that intra-axonal compartments are fundamental units for localization of multiple molecules, molecular mechanisms for generating or maintaining their compartmental localization could be diverse (Katsuki, 2009).

Another critical issue raised in the previous studies in vivo is how the intra-axonal localization of guidance receptors is maintained over time. If the guidance receptors are freely diffusible on the axonal membrane, they may spread along the axon, leading to a uniform distribution. FRAP experiments in cultured neurons revealed that localized receptors (ROBO3-EGFP and DRL-EGFP) are indeed mobile within the intra-axonal compartment. Although the mobility of these localized receptors across the compartment boundary was not directly measurable, the mobility of several transmembrane proteins (ROBO-EGFP and CD8-GFP) and lipid-anchored protein (GFPgpi) that distribute along the entire axon length was significantly restricted at the compartment boundary. This restriction is likely due to the diffusion barrier that spans over a 10 μm axon length around the boundary. It is proposed that this barrier is a part of the mechanisms that maintain the pattern of compartment-specific membrane proteins, as shown in different subcellular regions such as the tight junction of epithelial cells, the posterior ring of sperm, the cleavage furrow of dividing yeast and mammalian cells, and the initial segment of mammalian neurons. No significant barrier effect on GAP-GFP, which resides in the inner leaflet of the plasma membrane, was detected. It was also observed that vesicles containing membrane proteins pass through the barrier region. Thus, a model is favored in which the barrier becomes effective only after the molecules are inserted into the axonal membrane. It would be important to test whether or not a diffusion barrier exists in vivo, and whether or not it plays a role in the development of the nervous system (Katsuki, 2009).

An important but yet poorly explored question is the role of the guidance receptors localized on axon shafts. A straightforward explanation can be offered based on non-cell-autonomous functions of guidance receptors or membrane proteins in general; they may 'label' axon pathways through specific adhesion (fasciculation), or through presenting their ligands, thereby providing instructive spatial cues for the navigation of other axons. For example, Fasciclin cell-adhesion molecules have been suggested to provide pathway labels for guiding other growth cones. Drosophila Netrin receptor Frazzled/DCC relocates its ligand Netrin to strategic positions in the nervous system, thereby generating guidance information for a longitudinal pioneer neuron. Other studies reported that guidance receptors can also play non-cell-autonomous roles in cell migration and synaptogenesis. Thus, spatial patterns of molecules on axon shafts likely have direct roles in neuronal circuit formation (Katsuki, 2009).

Lastly, it is proposed that the compartmentalization of the axonal membrane could be a common basis for the structure and function of the nervous system. In the Drosophila ventral nerve cord, formation of the longitudinal axon tracts depends on the expression of ROBO receptors. On the other hand, longitudinal axon tracts are considered as the site for synapse formation, because synaptic proteins such as synaptotagmin and synapsin accumulate on the longitudinal tracts. This study found that in cultured neurons both ROBO receptors and synaptic proteins localize to the distal axon compartment. This may suggest that the spatial distribution of guidance molecules and synaptic proteins can be collectively governed by the compartmentalization of the axonal membrane. Future work to identify the molecular basis of the compartmentalization, and to establish the link between cellular identity and this intracellular pattern, will aid in determining how intra-axonal patterning contributes to tissue organization (Katsuki, 2009).

Protein Interactions

Frazzled (Fra) is the DCC-like Netrin receptor in Drosophila that mediates attraction; Roundabout (Robo) is a Slit receptor that mediates repulsion. Both ligands, Netrin and Slit, are expressed at the midline; both receptors have related structures and are often expressed by the same neurons. To determine if attraction versus repulsion is a modular function encoded in the cytoplasmic domain of these receptors, chimeras were created carrying the ectodomain of one receptor and the cytoplasmic domain of the other and their function in transgenic Drosophila was tested. Fra-Robo (Fra's ectodomain and Robo's cytoplasmic domain) functions as a repulsive Netrin receptor; neurons expressing Fra-Robo avoid the Netrin-expressing midline and muscles. Robo-Fra (Robo's ectodomain and Fra's cytoplasmic domain) is an attractive Slit receptor; neurons and muscle precursors expressing Robo-Fra are attracted to the Slit-expressing midline (Bashaw, 1999).

In Drosophila, the same midline cells normally secrete both Netrins and Slit. Growth cones can simultaneously respond to both ligands in a cell-specific fashion. Some growth cones express high levels of Fra and low levels of Robo, and they extend toward and across the midline. Other growth cones appear to express high levels of both receptors, and they can extend toward the midline, but they do not cross it. Growth cones can dramatically change their levels of Robo expression; once they cross the midline, growth cones increase their level of Robo, a change that prevents them from crossing the midline again. Such complex and dynamic behavior requires growth cones to be able to simultaneously respond to both attractants and repellents and to integrate these signals and respond to the relative balance of forces. Introducing a chimeric receptor into this finely tuned system leads to dramatic phenotypes. Adding a receptor that responds to Netrin as a repellent leads to a comm-like phenotype in which too few axons cross the midline. Adding a receptor that responds to Slit as an attractant leads to the opposite robo- or slit-like phenotypes, in which too many axons cross the midline or remain at the midline, respectively. These phenotypes are dose dependent, suggesting that by adding more chimeric receptor, the relative balance can be tipped and in tis way the growth cone's response is selectively controlled. This striking dosage sensitivity raises the possibility of using these phenotypes as the basis for genetic suppressor screens to identify signaling components that function downstream of attractive and repulsive guidance receptors (Bashaw, 1999).

Another finding of this study is that the signal transduction machinery for attraction and repulsion downstream of these receptors appears to be present in all neurons, and probably in all migrating muscle precursors as well. All neurons expressing either Fra-Robo or Robo-Fra appear to behave the same, regardless of their environment: if they express Fra-Robo, they stay away from the midline; if they express Robo-Fra, they extend toward the midline. No other factor appears to intrinsically commit one growth cone or another to only one kind of response. The same is true for migrating muscle precursors. Normally, many of them express Robo and migrate away from the Slit-expressing midline. However, given the opportunity (by transgenic expression of Robo-Fra), they clearly contain the full machinery for the opposite response. In all these transgenic experiments, the growth cone or muscle response always correlated with the level of receptor (Brashaw, 1999 and references).

The finding that the cytoplasmic sequence determines the response of a guidance receptor raises a number of interesting questions. Attraction might lead to a local change favoring actin polymerization over depolymerization, while repulsion might lead to the opposite change. But is guidance that simple? The cytoplasmic sequences of five different families of repulsive guidance receptors are now known: UNC-5s, Eph receptors, Neuropilins, Plexins, and Robos. Interestingly, they appear to share little if any sequence similarity to one another in their cytoplasmic domains. It is possible, of course, that they bind different adapter proteins that converge on the same repulsive motility machinery. But it is equally likely that not all repulsion is the same and that different classes of repulsive receptors mediate different types of responses in the growth cone. It could be that what is lumped together under the term 'repulsion' actually represents several molecularly distinct mechanisms that negatively influence local growth cone behavior. Just what these different cytoplasmic domains do, and how many different types of repulsion exist, awaits future investigation (Bashaw, 1999 and references).

Netrin is a secreted protein that can act as a chemotropic axon guidance cue. Two classes of Netrin receptor, DCC and UNC-5, are required for axon guidance and are thought to mediate Netrin signals in growth cones through their cytoplasmic domains. However, in the guidance of Drosophila photoreceptor axons, the DCC ortholog Frazzled is required not in the photoreceptor neurons but instead in their targets, indicating that Frazzled also has a non-cell-autonomous function. This study shows that Frazzled can capture Netrin and 'present' it for recognition by other receptors. Moreover, Frazzled itself is actively localized within the axon through its cytoplasmic domain, and thereby rearranges Netrin protein into a spatial pattern completely different from the pattern of Netrin gene expression. Frazzled-dependent guidance of one pioneer neuron in the central nervous system can be accounted for solely on the basis of this ability of Frazzled to control Netrin distribution, and not by Frazzled signaling. A model of patterning mechanism is proposed in which a receptor rearranges secreted ligand molecules, thereby creating positional information for other receptors (Hiramoto, 2000).

In vitro chemotropic responses of growth cones to Netrin indicate that graded distribution of Netrin may be important for guiding axons in vivo. A Netrin gradient could be produced by constant secretion followed by diffusion and degradation. However, in the ventral nerve cord of the Drosophila embryo the distribution of Netrin protein cannot be explained by such a mechanism. Drosophila Netrin is encoded by two genes, Netrin-A and Netrin-B. Although Netrin messenger RNA is abundant in the midline and the ventral region of the nerve cord, Netrin-A and Netrin-B proteins localize in the dorsolateral region, where no Netrin mRNA is detected. Even when Netrin-B transcription is artificially restricted to midline cells, Netrin-B still accumulates in the dorsolateral region as in wild-type embryos, rather than forming a gradient centered at the midline. This suggests that Netrin is either transported to the dorsolateral region or is selectively captured there after secretion (Hiramoto, 2000).

Frazzled is a good candidate for a molecule that relocalizes Netrin. Its accumulation is most evident on axon stalks of the commissural region, and its ortholog, DCC, is known to bind Netrin. Moreover, the dorsolateral Netrin-positive region precisely matches Frazzled distribution. In the absence of Frazzled, Netrin does not accumulate dorsolaterally and Netrin-B is observed only on cell bodies that express Netrin-B mRNA. Moreover, when Frazzled is misexpressed in ventral unpaired median (VUM) cells, ectopic Netrin-B protein is found on their surface even though these cells do not express Netrin-B. These data indicate that ectopic Frazzled can capture Netrin synthesized elsewhere, and suggest that Frazzled localizes Netrin in the dorsolateral region of ventral nerve cord. As expected, Frazzled distribution is unaltered in Netrin-A, Netrin-B double-mutant embryos (Hiramoto, 2000).

Frazzled itself is not found uniformly throughout the membrane, but is concentrated in specific regions of the axon, indicating that its distribution may also be regulated. Localized distribution within the neuron has been observed for Roundabout (Robo), a transmembrane receptor for another guidance molecule, Slit, and the localization signal of Robo has been mapped to its cytoplasmic or transmembrane domain. Similarly, Frazzled lacking its cytoplasmic domain (Fra-deltaC) is distributed throughout the cell membrane. Furthermore, Robo-Fra, a chimaera with the extracellular and transmembrane domain of Robo and the cytoplasmic domain of Frazzled, is distributed in the same way as full-length Frazzled. This shows that the cytoplasmic domain of Frazzled is necessary and sufficient for proper localization. Fra-Robo, a chimaera with the extracellular and transmembrane domain of Fra and the cytoplasmic domain of Robo, was also expressed in frazzled minus animals. In such embryos, the Fra-Robo fusion protein fails to distribute in the wild-type Frazzled pattern, and Netrin-B is mislocalized to many of the sites of Fra-Robo accumulation. These data show that Frazzled captures Netrin with its extracellular domain, whereas Frazzled distribution is controlled by a localization signal in the cytoplasmic domain (Hiramoto, 2000).

An investigation was carried out to see how axons are guided by the Netrin that is captured by Frazzled. Focused was placed on an identified pioneer neuron, dMP2, that requires Netrin-A/Netrin-B and frazzled function. dMP2 axons extend laterally and then turn posteriorly to form the initial longitudinal axon pathway. Precisely at the turning point, the medial edge of dorsolateral Netrin accumulation abuts the dMP2 pathway. dMP2 axons make pathfinding errors in both Netrin-A, Netrin-B double mutants and frazzled mutants, and such defects are often accompanied by severe disorganization of longitudinal tracts. These data may indicate that dMP2 axon guidance by Frazzled and Netrin is essential for the formation of the longitudinal axon pathway (Hiramoto, 2000).

To investigate how Frazzled functions in the guidance of dMP2, a test was performed to see whether frazzled is required in the dMP2 neuron itself. Contradictory to the idea that Frazzled is a Netrin sensor in dMP2 growth cones, Frazzled protein is not detected in dMP2. Moreover, expressing Frazzled in dMP2 in a frazzled minus background does not rescue the defects in dMP2 axon guidance. In contrast, when Frazzled is expressed in many central neurons in the frazzled minus background, the defects of dMP2 axon guidance are rescued, even though Frazzled is not expressed in dMP2. These data indicate that, for this guidance decision, Frazzled acts as a pathway marker and not as a sensor in growth cones (Hiramoto, 2000).

The ability of Frazzled to capture Netrin raises the possibility that Frazzled guides dMP2 by capturing and presenting Netrin to dMP2. To test this, Fra-deltaC was expresssed in a frazzled-mutant background to create an ectopic Netrin-B positive region near the axon pathway of dMP2 without changing the pattern of Netrin transcription. In such embryos, dMP2 growth cones spread abnormally over this surface of artificial Netrin accumulation. Also, Netrin-B was directly misexpressed in cell bodies located near the dMP2 axon pathway. Again, dMP2 growth cones respond to the ectopic Netrin-B-positive region. This strongly suggests that the response of dMP2 to the ectopic Frazzled extracellular domain is due to a response to the Netrin bound to the domain (Hiramoto, 2000).

An implication of these data is that dMP2 uses a Netrin receptor other than Frazzled to respond to Netrin. Redirection of dMP2 growth cones to ectopic Netrin indicates that Netrin is perceived as an attractive cue to dMP2. As the Drosophila genome does not contain any other genes with significant homology to DCC, it is expected that the Netrin receptor expressed in dMP2 is structurally different from the DCC class of Netrin receptors (Hiramoto, 2000).

These data indicate that Frazzled captures and rearranges Netrin, and presents it to other growth cones. The capture/relocation mechanism can create a precise Netrin distribution even in regions that are quite distant from the source of Netrin protein. Just as Frazzled presents Netrin to the dMP2 axon at its lateral turning point, the vertebrate Frazzled ortholog DCC also captures Netrins, and is localized to the point where the commissural axons turn longitudinally. Presentation of Netrin may thus be a general feature of DCC proteins. How Netrin reaches its final location is not yet clear. As Netrin-B does not localize to all Fra-Robo positive regions even when they are close to a source of Netrin-B , relocation of Netrin is likely to involve transport along axons rather than diffusion alone. Perhaps active relocalization of receptors such as Frazzled or Robo may be used to transport ligands to the final target area, where they are interpreted by other receptors. In addition to neuronal axons, extended cellular processes, such as the cytonemes of Drosophila imaginal discs and vertebrate limb buds, have been implicated in other patterning systems. It will be interesting to see whether such systems also use capture/relocation mechanisms to generate precise spatial patterns away from the source of the diffusible morphogen (Hiramoto, 2000).

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