The expression of sqz is largely restricted to subsets of cells in the CNS throughout embryonic and first instar larval (L1) development. Using sqzGAL4 to drive expression of the axonal reporter, UAS-τ-myc, sqz was found to be expressed in a population of lateral interneurons, primarily projecting axons in the anterior and posterior commissures. In sqz mutants, expressing neurons are generated and appear to extend axons along the appropriate tracts. Using both sqzlacZ and sqzGAL4, tests were performed for overlap with ap; sqz and ap were found to be co-expressed specifically within the thoracic ap cluster. Co-expression of sqz and ap is evident from the onset of ap expression at stage 14, with one neuron typically expressing higher levels of sqz. By stage 17, sqz expression is restricted to two neurons within the ap-cluster, with one neuron typically continuing to display higher levels of expression. Expression overlap between sqz and FMRFa was tested in late stage 17 embryos, when FMRFa expression commences; sqz is indeed selectively expressed at higher levels within the FMRFa Tv neuron. Thus, the six neurons within the VNC that co-express ap and higher levels of sqz selectively express the neuropeptide FMRFa and innervate the three specialized neuroendocrine glands -- the dorsal neurohemal organs (Allan, 2003).
In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, how each of these factors contributes to ap neuron diversity has been resolved. These factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, it was also found that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits (Allan, 2005).
Within every VNC hemisegment, ap is expressed by one dorsal neuron (dAp) and two ventral neurons (vAp). Additionally, in thoracic VNC hemisegments, ap is expressed by a lateral cluster of four neurons (the ap cluster), termed the Tv, Tvb, Tva, and Tvc neurons. These ap neurons are phenotypically diverse. The axons of most ap neurons project within an ipsilateral fascicle (ap fascicle) that projects to the brain, whereas the axons of the Tv cell exit the VNC at the midline to innervate the dorsal neurohemal organs (DNH). A subset of ap neurons is peptidergic (the Tv, Tvb, and dAp neurons). As is characteristic for the vast majority of Drosophila peptidergic neurons, these cells express high levels of the peptide biosynthetic enzyme peptidylglycine alpha-hydroxylating monooxygenase (PHM). However, this peptidergic subset is also diverse: Tv cells selectively express the dFMRFa neuropeptide, whereas Tvb and dAp cells selectively coexpress three peptide biosynthetic enzymes -- PC2, Furin1, and PAL2 -- although the identity of their secreted neuropeptide(s) remains unknown. This coexpression in Tvb and dAp cells suggested a functional grouping and a common name, 'Ap-let' cells. For clarity, the ap neurons will be considered as three classes: (1) Tv cells express dFMRFa and PHM and innervate the DNH; (2) Ap-let (Tvb and dAp) cells express PHM, PC2, Furin1, and PAL2; (3) the vAp, Tva, and Tvc cells are nonpeptidergic (Allan, 2005).
ap, sqz, dimm, and the BMP pathway act in a combinatorial code to regulate dFMRFa in the Tv cell (ap, sqz, dimm, and the BMP pathway) and furin1 (ap, dimm) in Ap-let cells. Importantly, however, each regulator also plays critical roles within these ap neurons independent of the other regulators. Ap independently acts to regulate axon pathfinding by all ap cells except the Tv. Dimm independently controls PHM in the Tv and Ap-let cells. Moreover, Sqz independently acts via the N pathway to regulate cell identity within the ap cluster, upstream of both Ap and Dimm, apparently by suppressing the Tvb cell fate to favor the Tv fate. The Ap-let cells do not express Sqz, nor do they have an activated BMP pathway. In these neurons, Ap activates the expression of Dimm, and both act together to activate the expression of the peptide-processing enzyme Fur1. The Tva and Tvc cells of the ap cluster do not express Dimm and do not have an activated BMP pathway. Remarkably, the differences inferred between regulatory circuits for the two classes of peptidergic cells are highly reminiscent of differences in regulatory circuits that operate during the differentiation of distinct noradrenergic neurons. Together, these sets of studies support the proposition that epistatic relations between regulators underlying the production of a common phenotype may differ according to cell type (Allan, 2005).
The loss-of-function and gain-of-function phenotypes presented for ap, sqz, dimm, and the BMP pathway, suggest that they act in a combinatorial fashion to regulate dFMRFa expression in the Tv neuron. Likewise, the results indicate that ap and dimm, in the absence of sqz and the BMP pathway, combine to activate Fur1 in the Ap-let neurons, Tvb and dAp. In order to determine whether these regulators act simultaneously on dFMRFa and Fur1, rather than in a genetic hierarchy, the epistatic and biochemical relationship between these regulators were studied. Only one clear epistatic relationship was found; Ap activates the expression of Dimm in the majority of ap neurons. Therefore, it was important to determine whether Dimm acted downstream of Ap to independently and more directly regulate dFMRFa and Fur1 expression. This hypothesis was tested in two complementary tests. (1) Rescuing Dimm function in ap neurons that were absent for Ap function, yielded a nearly complete rescue of dFMRFa in Tv neurons, but only relatively weak rescue of Fur1 in Ap-let neurons. (2) Panneuronal co-misexpression of both ap and dimm triggers ectopic dFMRFa expression in a much greater number of neurons than does either regulator alone. These data indicate that Dimm functions together with Ap to achieve wild-type levels of dFMRFa and, more notably, Fur1. Thus, ap and dimm appear to display both hierarchical and combinatorial interactions. This hypothesis has precedent in studies of the developing pancreas, in which Foxa2 is required for pdx-1 transcription in β cells and later interacts directly with PDX-1 protein to regulate target gene expression, including maintained pdx-1 expression. Biochemical data are also consistent with the possibility that a combinatorial Ap, Dimm, and Sqz code that activates dFMRFa and dFur1 involves direct protein interactions. These may exist within larger complexes bridged by Chip, since Dimm can interact directly with both Ap and Chip, and this in turn may explain why Dimm partially rescues both the ap mutant dFMRFa and Fur1 phenotypes. These multiple interactions are reminiscent of synergistic interactions suggested between mammalian bHLH proteins, LIM-HD proteins, and the Chip homolog, LDB1/NLI. The simplest explanation for restricted patterns of neuropeptides and certain neuropeptide biosynthetic enzymes features a combinatorial hypothesis. More specifically, it is proposed that different combinatorial codes of transcription factors act cell specifically to effect differing patterns of neuropeptides and associated processing enzymes (Allan, 2005).
Ap expression is an early marker of ap cell differentiation, and it is required for proper axonal pathfinding by most ap neurons, although not by the Tv cell. In contrast, neither Sqz nor Dimm appear to control ap cell morphogenesis. An independent role for Sqz occurs early in ap cell differentiation, at a time when postmitotic cell fates are being determined. It is surprising that such cell fate changes can be rescued by UAS-Dl. Why would the frequently used N pathway signaling system depend upon a much more restricted regulator like sqz for proper activity? Increasing evidence points to major mechanistic differences between N signaling during neuroblast specification and during asymmetric division, where asymmetric divisions specifically require neuralized, numb, and sanpodo. No expression of sqz is found in neuroblasts, but expression is evident in many VNC cells. Therefore, it is proposed that factors like Sqz coordinate late N signaling with cell specification and/or cell cycle genes (Allan, 2005).
Dimm acts independently of Ap, Sqz, and the BMP pathway to activate expression of the neuropeptide-processing enzyme PHM. The evidence regarding the independent role of Dimm suggests that it is a master regulator of neuroendocrine cell fate. dimm expression is highly correlated with a neuroendocrine/peptidergic cellular identity, where it regulates the expression of almost all neuropeptides and their processing enzymes examined to date, especially within those neurons that express peptides that are processed to include an α-amidated C terminus. This is a significant cellular pattern, because more than 90% of Drosophila neuropeptides are amidated. Furthermore, high-level expression of the PHM enzyme is absolutely required for amidation and serves as an excellent marker for most peptidergic neurons in Drosophila. Finally, PHM expression appears to be dedicated to neuroendocrine peptide biosynthesis; it is exclusively found within the luminal domain of secretory vesicles. Thus, PHM expression provides a faithful marker for the peptidergic/neuroendocrine cell fate. This study has shown that PHM is dominantly induced by dimm overexpression throughout most or all of the CNS. This evidence, together with the loss-of-function data argues strongly that dimm is a neuroendocrine master regulator, with properties akin to those of other bHLH proteins in regulating cell fate (Allan, 2005).
As anticipated, more restricted peptidergic traits such as dFMRFa and Fur1 expression are dependent upon combinatorial codes. Importantly, however, the selection of cell-specific peptidergic markers arises from a deterministic interaction between a peptidergic master regulator and a cell-specific combinatorial code. There exists a clear analogy between the action of dimm in developing neurons and results regarding the glial cells missing (gcm) gene. Studies have shown that gcm is both necessary and sufficient for glial cell specification within the DrosophilaVNC. gcm is able to ectopically activate generic glial genes, such as reversed polarity, and also activates subclass-specific glial genes, but only in certain prescribed subsets of cells. Thus, similar to gcm, it is predicted that dimm is a master regulator of core neuroendocrine genes in most peptidergic/neuroendocrine cells. It will be of interest to determine which genes beyond PHM are under dimm control. In parallel, dimm combines with local-acting factors to help activate subclass-specific genes (e.g., neuropeptide-encoding genes) within peptidergic cell subsets (Allan, 2005).
The genes studied here combine to regulate dFMRFa and Fur1 but also have independent roles within the same cells. This raises the issue of how Dimm, for instance, can complex with Ap/Sqz on dFMRFa and also act independently on PHM within the same nucleus. Surprisingly, no clear evidence of an antagonistic relationship between the individual roles of Ap, Sqz, and Dimm was found. For example, co-misexpression of ap with dimm does not obviously suppress the ectopic PHM expression observed when dimm alone is misexpressed. Likewise, misexpression of sqz in the Fur1-expressing dAp/Tvb cells does not suppress Fur1. Thus, it appears that the independent mechanisms of regulator action are robust and can coexist with combinatorial functions. Therefore, it is proposed that these regulators operate within a bistable organizational mechanism. With respect to independent roles, it is proposed that Dimm operates independently of Ap and Sqz to dominantly induce specific target genes (e.g., PHM) within all neuronal lineages by forming heterodimers with a class A bHLH like Da, or by forming homodimers. The Drosophila bHLH Twist protein has distinct regulatory roles in vivo, acting either as a heterodimer with Da, or as a homodimer. Notably, the mammalian ortholog of Dimm, Mist1, forms functional homodimers to promote the differentiation of pancreatic secretory cells (Allan, 2005).
The TGFβ/BMP signal transduction pathway plays critical roles during a number of developmental events, and mutants affecting the Drosophila BMP pathway show dramatic defects in embryonic development. In contrast, in the Tv neuron, BMP signaling plays a much more subtle role, and although it is critical for dFMRFa expression, no effects were found upon the expression of sqz, ap, or dimm or on the general peptidergic marker PHM in wit mutants. Although these studies cannot rule out other roles for the BMP pathway in Tv neurons, it is tempting to speculate that target-derived BMP signaling in neurons may have quite a limited set of nuclear readouts in each specific neuronal subclass (Allan, 2005).
Proper information processing in neural circuits requires establishment of specific connections between pre- and post-synaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).
Neural stem cell quiescence is an important feature in invertebrate and mammalian central nervous system development, yet little is known about the mechanisms regulating entry into quiescence, maintenance of cell fate during quiescence, and exit from quiescence. Drosophila neural stem cells (neuroblasts or NBs) provide an excellent model system for investigating these issues. Drosophila NBs enter quiescence at the end of embryogenesis and resume proliferation during larval stages; however, no single neuroblast lineage has been traced from embryo into larval stages. This study establishes a model NB lineage, NB3-3, which allows reproducibly observation of lineage development from NB formation in the embryo, through quiescence, to the resumption of proliferation in larval stages. Using this new model lineage, a continuous sequence of temporal changes is shown in the NB, defined by known and novel temporal identity factors, running from embryonic through larval stages; quiescence suspends but does not alter the order of neuroblast temporal gene expression. NB entry into quiescence is regulated intrinsically by two independent controls: spatial control by the Hox proteins Antp and Abd-A, and temporal control by previously identified temporal transcription factors and the transcription co-factor Nab (Tsuji, 2008).
This study has revealed for the first time the temporal changes in a Drosophila NB lineage from embryonic NB formation, through entry into quiescence, to resumption of proliferation in larval stages. Using a model NB system with which the complete lineage formation can be reproducibly traced at the resolution of individual cell divisions, it was shown that despite considerable differences in extracellular environment the temporal changes (as defined by the switching of transcription factor/co-factor expression) proceeded continuously in each NB throughout the embryonic and larval stages. Moreover, mutual regulation was found between quiescence and the series of the temporal transcription factors/co-factor; the temporal transcription factors/co-factor endogenously control the timing of triggering NB quiescence, which in turn suspends the switching of late temporal transcription factor expression (Tsuji, 2008).
In the Antp mutant and following ectopic expression of Abd-A there was a lack of NB quiescence, and consequently what appeared to be a precocious generation of larval neurons during embryogenesis was observed. This strongly supports the notion that temporal changes in NBs actually continue in sequence before and after quiescence, i.e., through embryogenesis and larval stages, and in the absence of quiescence the changes occur precociously. In addition, this indicates that spatial and temporal factors control NB quiescence through independent routes (Tsuji, 2008).
Antp mutants did not exhibit NB3-3T quiescence in all thoracic T1-T3 segments. In Antp mutants, epidermis in T2 and T3 segments transform into that in the T1 segment, and some thoracic NB lineages retain thoracic-specific features. These facts indicate that the inhibition of NB3-3T quiescence by Antp mutation is not just a consequence of global transformation of thoracic-to-abdominal segments but rather results from specific effects on individual NBs. NB-specific misexpression of Abd-A also repressed Antp and inhibited NB3-3T quiescence. This also provides evidence that the effect is specific to NBs. Furthermore, because the effect could be observed even when Abd-A was induced after several divisions of the NB, thoracic NBs probably maintain plasticity of their identities during lineage formation (Tsuji, 2008).
It was shown that the temporal transcription factors/co-factor Pdm, Cas, Sqz and Nab play a role in triggering NB quiescence intrinsically in NBs. All of these factors also controlled temporal specification within late lineages of embryonic NBs in both thoracic and abdominal segments. This was confirmed by further examining the relationships of the temporal factors. For example, the loss of Pdm function in NB3-3T resulted in precocious transcription factor switching and precocious quiescence. Conversely, in cas mutant embryos, in which Pdm expression was de-repressed, quiescence was inhibited and expression of late-stage-specific temporal factors disappeared. Similar to Pdm upregulation, loss of nab function resulted in loss of both transcription factor switching and quiescence (Tsuji, 2008).
Although Nab and Sqz can form a complex, nab and sqz mutants displayed very different phenotypes. Both mutants showed de-repression of Kr expression; however, sqz mutants showed no other abnormality in transcription factor switching, whereas nab mutants showed the above-mentioned defects in transcription factor switching and timing of quiescence. These mutant phenotypes revealed that regulation of late temporal events is distributed into multiple pathways. Pdm probably coordinately regulates all factors involved in the timing of NB quiescence, because the loss of Pdm alone is sufficient to cause precocious entry into quiescence (Tsuji, 2008).
Nab and Sqz were shown to work for NB quiescence in NBs. The Nab/Sqz-mediated repression of Kr may be controlled in NBs due to changes in NB temporal identity, or in both NBs and their neurons. Nab might inhibit transcription by recruiting the nucleosome remodeling and deacetylase chromatin remodeling complex as does mammalian Nab (Srinivasan, 2006). Mammalian Nab acts with EGR-1, EGR-2 to determine the fate of cells in hematopoiesis (Laslo, 2006; Svaren, 1996), but whether it can act with the mammalian homolog of LIN-29/Sqz has not been reported. Loss of lin-29, a C. elegans homolog of sqz, causes a heterochronic phenotype in which adulthood is not reached and molting is repeated (Ambros, 1984; Rougvie, 1995). C. elegans has a nab homolog gene, mab-10, that acts with lin-29 in a heterochronic genetic cascade (Tsuji, 2008).
It is unclear what molecular mechanisms enable NBs to suspend the switching of transcription factor expression and maintain temporal identity during quiescence. It is known that the mechanisms for switching expression of early temporal transcription factors can be either cell division dependent or independent. Irrespective of the mechanism used, a NB can 'memorize' its temporal state before quiescence and resume the intrinsic temporal changes once cell cycle progression is reactivated. Embryonic stem cells may maintain multipotency during a slow proliferation state by staying in S phase. When quiescent NBs re-entered the cell cycle, their initial progeny incorporated BrdU fed since hatching, indicating that quiescent NBs stay either prior to S phase or early in S phase. It will be important to identify the point in the cell cycle at which NB enters quiescence (Tsuji, 2008).
Another well-established mechanism that governs temporal aspects of lineage formation is the heterochronic gene cascade in C. elegans. This cascade contains one each of the hunchback homolog and lin-29 genes and generates five distinct temporal cell identities within a single cell lineage. Drosophila NB lineage formation uses two types of Zn-finger proteins, namely the Hb/Cas class [Cas shares DNA-binding characteristics with Hb and the Kr/LIN-29 class. These pairs are expressed three times in NB lineages in the following order: (1) Hb and Kr-> (2) Cas, Kr and Sqz--> Cas and DmLin-29-->end of lineage. This sequence seems to produce at least ten distinct temporal identities within an NB lineage. The repetitive use of these temporal transcription factors in three distinct phases appears to have made the NB lineage longer and more diverse. Lack of Hb also generates NB lineage variety; the NB3-3 and NB2-1 lineages lack Hb and initiate their lineage with Kr. Although the model NB employed in this study lacks Hb, the sequence and entry into quiescence described in this study are common to many typical NB lineages that start with Hb. Interesting questions from the perspective of evolution are how do the three phases combine to form a single lineage and how has NB quiescence evolved in the middle of the NB lineages (Tsuji, 2008)?
Neural stem cells in the mouse cerebral cortex go through ~11 divisions and some enter quiescence in late embryo. The possibility has to be considered that mammalian neural stem cell and Drosophila NB share a similar intrinsic mechanism that induces quiescence (Tsuji, 2008).
Search PubMed for articles about Drosophila Squeeze
Allan, D. W., St Pierre, S. E., Miguel-Aliaga, I. and Thor, S. (2003). Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code. Cell 113(1): 73-86. Medline abstract: 12679036
Allan, D. W., Park, D., St. Pierre, S. E., Taghert, P. H. and Thor, S. (2005). Regulators acting in combinatorial codes also act independently in single differentiating neurons. Neuron 45: 689-700. Medline abstract: 15748845
Ambros, V. and Horvitz, H. R. (1984). Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226: 409-416. PubMed Citation: 6494891
Berman, B. P., et al. (2004). Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura. Genome Biol. 5(9): R61. Medline abstract: 15345045
Clements, M., Duncan, D. and Milbrandt, J. (2003). Drosophila NAB (dNAB) is an orphan transcriptional co-repressor required for correct CNS and eye development. Dev. Dyn. 226: 67-81. Medline abstract: 12508226
Herrero, P., et al. (2007). Squeeze involvement in the specification of Drosophila leucokinergic neurons: Different regulatory mechanisms endow the same neuropeptide selection. Mech. Dev. 124: 427-440. Medline abstract: 17442544
Felix, J. T., Magarinos, M. and Diaz-Benjumea, F. J. (2007). Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. Development 134(10): 1845-52. Medline abstract: 17428824
Harris, D. T. and Horvitz, H. R. (2011). MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C. elegans. Development 138(18): 4051-62. PubMed Citation: 21862562
Komiyama, T. and Luo, L. (2007). Intrinsic control of precise dendritic targeting by an ensemble of transcription factors. Curr. Biol. 17(3): 278-85. Medline abstract: 17276922
Laslo, P., Spooner, C. J., Warmflash, A., Lancki, D. W., Lee, H. J., Sciammas, R., Gantner, B. N., Dinner, A. R. and Singh, H. (2006). Multilineage transcriptional priming and determination of alternate hematopoietic cell fates. Cell 126: 755-766. PubMed Citation: 16923394
Le, N., Nagarajan, R., Wang, J. Y., Svaren, J., LaPash, C., Araki, T., Schmidt, R. E. and Milbrandt, J. (2005). Nab proteins are essential for peripheral nervous system myelination. Nat. Neurosci. 8: 932-940. Medline abstract: 16136673
Mechta-Grigoriou, F., Garel, S. and Charnay, P. (2000). Nab proteins mediate a negative feedback loop controlling Krox-20 activity in the developing hindbrain. Development 127: 119-128. Medline abstract: 10654606
Rougvie, A. E. and Ambros, V. (1995). The heterochronic gene lin-29 encodes a zinc finger protein that controls a terminal differentiation event in Caenorhabditis elegans. Development 121: 2491-2500. PubMed Citation: 7671813
Sevetson, B. R., Svaren, J. and Milbrandt, J. (2000). A novel activation function for NAB proteins in EGR-dependent transcription of the luteinizing hormone beta gene. J. Biol. Chem. 275: 9749-9757. Medline abstract: 10734128
Srinivasan, R., Mager, G. M., Ward, R. M., Mayer, J. and Svaren, J. (2006). NAB2 represses transcription by interacting with the CHD4 subunit of the nucleosome remodeling and deacetylase (NuRD) complex. J. Biol. Chem. 281: 15129-15137. PubMed Citation: 16574654
Svaren, J., Sevetson, B. R., Apel, E. D., Zimonjic, D. B., Popescu, N. C. and Milbrandt, J. (1996). NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli. Mol. Cell. Biol. 16: 3545-3553. PubMed Citation: 8668170
Svaren, J., Sevetson, B. R., Golda, T., Stanton, J. J., Swirnoff, A. H. and Milbrandt, J. (1998). Novel mutants of NAB corepressors enhance activation by Egr transactivators. EMBO J. 17: 6010-6019. Medline abstract: 9774344
Swirnoff, A. H., Apel, E. D., Svaren, J., Sevetson, B. R., Zimonjic, D. B., Popescu, N. C. and Milbrandt, J. (1998). Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain. Mol. Cell. Biol. 18: 512-524. Medline abstract: 9418898
Tsuji, T., Hasegawa, E. and Isshiki, T. (2008). Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors. Development 135(23): 3859-69. PubMed Citation: 18948419
date revised: 15 December 2011
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