Gene name - pebbled
Synonyms - hindsight
Cytological map position - 4C7--4C15
Function - transcription factor
Symbol - peb
FlyBase ID: FBgn0001209
Genetic map position - 1-7.
Classification - zinc finger protein
Cellular location - nuclear
|Recent literature||Baechler, B. L., McKnight, C., Pruchnicki, P. C., Biro, N. A. and Reed, B. H. (2015). Hindsight/RREB-1 functions in both the specification and differentiation of stem cells in the adult midgut of Drosophila. Biol Open [Epub ahead of print]. PubMed ID: 26658272
The adult Drosophila midgut is established during the larval/pupal transition from undifferentiated cells known as adult midgut precursors (AMPs). Four fundamental cell types are found in the adult midgut epithelium: undifferentiated intestinal stem cells (ISCs) and their committed daughter cells, enteroblasts (EBs), plus enterocytes (ECs) and enteroendocrine cells (EEs). Using the Drosophila posterior midgut as a model, the function of the transcription factor Hindsight (Hnt)/RREB-1 was studied and its relationship to the Notch and Egfr signaling pathways. hnt wash shown to be required for EC differentiation in the context of ISC-to-EC differentiation, but not in the context of AMP-to-EC differentiation. In addition, hnt is required for the establishment of viable or functional ISCs. Overall, these studies introduce hnt as a key factor in the regulation of both the developing and the mature adult midgut. It is suggested that the nature of these contextual differences can be explained through the interaction of hnt with multiple signaling pathways.
|Deady, L. D., Li, W. and Sun, J. (2017). The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles. Elife 6. PubMed ID: 29256860
Follicle rupture, the final step in ovulation, utilizes conserved molecular mechanisms including matrix metalloproteinases (Mmps), steroid signaling, and adrenergic signaling. It is still unknown how follicles become competent for follicle rupture/ovulation. This study identified a zinc-finger transcription factor Hindsight (Hnt) as the first transcription factor regulating follicle's competency for ovulation in Drosophila. Hnt is not expressed in immature stage-13 follicle cells but is upregulated in mature stage-14 follicle cells, which is essential for follicle rupture/ovulation. Hnt upregulates Mmp2 expression in posterior follicle cells (essential for the breakdown of the follicle wall) and Oamb expression in all follicle cells (the receptor for receiving adrenergic signaling and inducing Mmp2 activation). Hnt's role in regulating Mmp2 and Oamb can be replaced by its human homolog Ras-responsive element-binding protein 1 (RREB-1). These data suggest that Hnt/RREB-1 plays conserved role in regulating follicle maturation and competency for ovulation.
|Farley, J. E., Burdett, T. C., Barria, R., Neukomm, L. J., Kenna, K. P., Landers, J. E. and Freeman, M. R. (2018). Transcription factor Pebbled/RREB1 regulates injury-induced axon degeneration. Proc Natl Acad Sci U S A. PubMed ID: 29295933
Genetic studies of Wallerian degeneration have led to the identification of signaling molecules (e.g., dSarm/Sarm1, Axundead, and Highwire) that function locally in axons to drive degeneration. This study identified a role for the Drosophila C2H2 zinc finger transcription factor Pebbled [Peb, Ras-responsive element binding protein 1 (RREB1) in mammals] in axon death. Loss of Peb in Drosophila glutamatergic sensory neurons results in either complete preservation of severed axons, or an axon death phenotype where axons fragment into large, continuous segments, rather than completely disintegrate. Peb is expressed in developing and mature sensory neurons, suggesting it is required to establish or maintain their competence to undergo axon death. peb mutant phenotypes can be rescued by human RREB1, and they exhibit dominant genetic interactions with dsarm mutants, linking peb/RREB1 to the axon death signaling cascade. Surprisingly, Peb is only able to fully block axon death signaling in glutamatergic, but not cholinergic sensory neurons, arguing for genetic diversity in axon death signaling programs in different neuronal subtypes. These findings identify a transcription factor that regulates axon death signaling, and peb mutant phenotypes of partial fragmentation reveal a genetically accessible step in axon death signaling.
|Lo, P. K., Huang, Y. C., Corcoran, D., Jiao, R. and Deng, W. M. (2019). Drosophila chromatin assembly factor 1 p105 and p180 subunits are required for follicle cell proliferation via inhibiting Notch signaling. J Cell Sci. PubMed ID: 30630896
Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. This study reports another side of CAF1 wherein the subunits CAF1-p105 and CAF1-p180 inhibit expression of Notch target genes and shows this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt), leading to Cut downregulation and inhibition of follicle cell mitosis. These studies further show Notch is functionally responsible for these phenotypes observed in CAF1-p105/p180-deficient follicle cells. Moreover, this study reveals that CAF1-p105/p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative feedback regulatory loop between Cut and Hnt underlying CAF1-p105/p180 regulation, which is crucial for follicle cell differentiation. In conclusion, these studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner.
Pebbled, also known as Hindsight, is involved in the process of germ band retraction. A short review of the significance of the germ band and the process of germ band closure is given below, before describing the role of Pebbled.
The layered, invaginated ventral area of the embryo that has developed by the time gastrulation is complete is referred to as the germ band. It gives rise to the germ layers (ectoderm and mesoderm), (not to be confused with germ line stem cells that give rise to egg and sperm). Germ band retraction (illustrated with on-line images ) is a process that shortens the germ band, following germ band extention in Drosophila. Beginning about 7 hours and 20 minutes after egg laying, germ band retraction is accompanied by the transition from a parasegmental to a segmental division of the embryo. During the shortening process, the amnioserosa spreads out from its compressed state to cover the whole of the dorsal surface. In the process of segmentation, deep ventral-lateral grooves form, corresponding to the incipient segmental boundaries. The interior aspects of these segmental boundaries are the sites for future muscle attachment.
During germ-band retraction, the packing of cells along the dorsal-ventral axis is altered; the dimensions of a segment are transformed from approximately 15-17 cells wide and 35 cells high to 13-15 cells wide and about 40 cells high. This small change in cell numbers across the segment cannot account for the observed change in overall dimensions, an increase of almost 100% in the dorsal-ventral axis and a reduction of about 50% in the anterior-posterior axis. It is most likely that germ-band shortening is primarily driven by changes in cell shape and packing, rather than by changes in the number or rearrangement of cells (Martinas Arias, 1993).
Another process that takes place progressively during germ band retraction is known as dorsal closure. It takes about two hours, beginning 11 hours after the start of development. Dorsal closure is the process whereby the stretched amnioserosa is covered by epidermal cells that will ultimately fuse at the dorsal midline. Genes involved in dorsal closure include rho, hemipterous and basket. Dorsal closure may be likened to the gathering shut of the opening in a string purse, in which cytoskeletal changes (contraction of the sub-membrane, cortical cytoskeleton) drive changes in cell shape, narrowing and lengthening cells of the epidermis, bringing them over the amnioserosa and then closing the epidermis over this "gathered together" cell layer. With its part in development played, the enclosed amnioserosa is then absorbed by the yolk.
Germ band retraction involves a number of genes including the Epidermal growth factor receptor, the Drosophila homolog of the mammalian insulin receptor (encoded by the inr gene), tailup, u-shaped, serpent and pebbled, the subject of this overview. Four of these genes (Egfr, peb, u-shaped and srp) are required for the maintenance of the amnioserosa. Consequently it is argued that peb expression in the amnioserosa is crucial for germ-band retraction. Two models for the involvement of the amnioserosa in germ-band retraction have been examined: a physical model suggests that the differentiated amnioserosa controls retraction throught direct physical interaction with cells of the germband. A chemical model suggests that maintenance of the amnioserosa produces or activates a signal that is received by the germ band and coordinates germ-band retraction (Yip, 1997).
Since Pebbled is expressed in the midgut, it became important to show whether this expression is important in driving germ band retraction. The embryonic midgut can be eliminated without affecting germ-band retraction. However, elimination of the amnioserosa results in the failure of germ-band retraction, implicating amnioserosal expression of Pebbled as crucial to this process. Ubiquitous expression of pebbled in the early embryo rescues germ-band retraction without producing dominant gain-of-function defects, suggesting that pebbled's role in germ-band retraction is permissive rather than instructive (Yip, 1997).
What then is the role of the amnioserosa in germ band retraction? Several lines of evidence suggest that the amnioserosa produces or activates signals that coordinate the morphogenetic alterations in the adjacent ectoderm during germ-band retraction. Thus the evidence favors a chemical signaling model. Both the Egfr and the Inr are expressed throughout the embryo with the exception that the Inr is never present in the amnioserosa and the Egfr is absent from the amnioserosa after stage 10. Based on these expression patterns and the fact that the products of these two genes are transmembrane receptor tyrosine kinases, it is possible that coordinating signals from the amnioserosa are received in the ectoderm by the Inr and/or the Egfr and are transduced into the shape changes and local cell rearrangements that drive germ-band retraction. The coordinating signals produced by the amnioserosa could be an activity or activites that process or activate the ligands for the receptor tyrosine kinases. Or these signals could function more indirectly through effects on the extracellular matrix (Yip, 1997). Pebbled is a nuclear zinc finger protein, suggesting that Hnt acts as a transcription factor regulating some aspect of amnioserosal signalling to surrounding ectoderm promoting germ band retraction.
Basket plays a key role in regulating the morphogenetic process of dorsal closure, which also serves as a model for epithelial sheet fusion during wound repair. During dorsal closure the JNK signaling cascade in the dorsal-most (leading edge) cells of the epidermis activates the AP-1 transcription factor comprised of Jun and Fos that, in turn, upregulates the expression of the dpp gene. Dpp is a secreted morphogen that signals lateral epidermal cells to elongate along the dorsoventral axis. The leading edge cells contact the peripheral cells of a monolayer extraembryonic epithelium, the amnioserosa, which lies on the dorsal side of the embryo. Focal complexes are present at the dorsal-most membrane of the leading edge cells, where they contact the amnioserosa. The JNK signaling cascade is initially active in both the amnioserosa and the leading edge of the epidermis. JNK signaling is downregulated in the amnioserosa, but not in the leading edge, prior to dorsal closure. The subcellular localization of Fos and Jun is responsive to JNK signaling in the amnioserosa: JNK activation results in nuclear localization of Fos and Jun; the downregulation of JNK signaling results in the relocalization of Fos and Jun to the cytoplasm. The Hindsight Zn-finger protein and the Puckered (Puc) JNK phosphatase are essential for downregulation of the JNK cascade in the amnioserosa. Persistent JNK activity in the amnioserosa leads to defective focal complexes in the adjacent leading edge cells and to the failure of dorsal closure. Thus focal complexes are assembled at the boundary between high and low JNK activity. In the absence of focal complexes, miscommunication between the amnioserosa and the leading edge may lead to a premature 'stop' signal that halts dorsalward migration of the leading edge. Spatial and temporal regulation of the JNK signaling cascade may be a general mechanism that controls tissue remodeling during morphogenesis and wound healing (Reed, 2001).
Expression of the Hnt Zn-finger transcription factor in the amnioserosa, particularly in those cells that abut the leading edge of the epidermis, is essential for this morphogenetic process. Hnt function has been shown in this study to be necessary for dorsal closure. A subset of hnt mutant embryos carrying the embryonic lethal alleles hnt704a and hntXO01 successfully complete germ band retraction but do not hatch. Analyses of cuticle preparations have revealed that 60% of hnt704a and 79% of hntXO01 embryos that complete retraction exhibit an anterior-open or dorsal-hole phenotype characteristic of the failure of dorsal closure (Reed, 2001).
hnt and JNK signaling pathway mutants interact genetically. hnt308 single mutants exhibited 41% embryonic lethality. When the dose of the JNK-encoding gene, basket (bsk), is reduced in a hnt308 mutant background (embryonic lethality was suppressed approximately 2-fold). These results suggest that, in hnt mutants, JNK signaling is upregulated (i.e., that the function of Hnt in dorsal closure is to downregulate JNK signaling). Thus, the reduction of JNK function is able to partially suppress the dorsal closure defect in hnt308 mutants (Reed, 2001).
To further test the role of Hnt in regulating JNK signaling, genetic interactions between hnt308 and dpp mutants were examined. A 50% reduction of dpp gene dose led to an 8-fold reduction in hnt308 embryonic lethality (5% versus 41%). Conversely, increasing the dose of the wild-type dpp gene from two to three copies led to a 2-fold increase in embryonic lethality (80% versus 41%). Examination of the hnt308 embryos carrying three doses of dpp revealed that the frequency of embryos with germ band retraction defects had doubled (41% as compared to 20%). These results provide the first evidence that Hnt may regulate both germ band retraction and dorsal closure through the JNK/DPP signaling pathways. The direction of the genetic interaction between hnt308 and dpp is consistent with the hypothesized role of Hnt to downregulate JNK signaling (Reed, 2001).
During normal development, JNK activity is downregulated in the amnioserosa prior to dorsal closure. Hnt is expressed in the amnioserosa, but not in the epidermis of the embryo. Given the genetic interactions between hnt and JNK pathway mutants, it was therefore asked whether JNK signaling occurs in the amnioserosa during normal embryogenesis. JNK signaling is shown to initially be present in the amnioserosa but it is is downregulated prior to dorsal closure (Reed, 2001).
The transcriptional activation of the genes dpp and puc provides a readout of JNK signaling activity in the leading edge. Enhancer trap lines dpplacZ and puclacZ were used as reporters for the activation state of the JNK pathway in the amnioserosa. These enhancer trap lines are expressed in the amnioserosa prior to germ band retraction. Toward the end of germ band retraction, JNK activity, as assayed by puclacZ and dpplacZ, decreases in the interior of the amnioserosa but persists in the amnioserosa perimeter cells that abut the leading edge. By the onset of dorsal closure, when JNK activity becomes elevated in the leading edge, the amnioserosa perimeter cells lose JNK activity, and there is reduced dpplacZ or puclacZ expression throughout the amnioserosa. It should be noted that perdurance of ß-galactosidase protein in the amnioserosa means that these analyses of the timing of loss of puclacZ and dpplacZ expression define the latest point in development at which JNK signaling is downregulated, not when such downregulation initiates. It is concluded that JNK signaling occurs in the amnioserosa prior to and during germ band retraction but is downregulated at or before the initiation of dorsal closure (Reed, 2001).
DJUN is activated through phosphorylation by JNK, and although it is capable of forming transcriptional activation complexes through homodimerization, it also forms heterodimers with Fos. Jun/Fos heterodimers belong to the AP-1 class of transcription factor complexes, are more stable than Jun homodimers, and are thought to be the biologically relevant protein complex (Reed, 2001).
To further investigate JNK signaling in the amnioserosa during dorsal closure, the expression of Jun and Fos were examined. In wild-type embryos, Jun and Fos accumulate at high levels in the amnioserosa prior to dorsal closure. During dorsal closure, Jun and Fos levels are highest in the leading edge but persist in the amnioserosa. In the amnioserosa, Jun and Fos are strictly nuclear prior to germ band retraction. Strikingly, both proteins begin to accumulate in the cytoplasm as germ band retraction is completed. While Fos becomes nearly exclusively cytoplasmic, Jun can be detected in both the cytoplasm and the nuclei during dorsal closure (Jun is present in a punctate pattern in the cytoplasm) (Reed, 2001).
To determine whether nuclear restriction of Jun and Fos is dependent on JNK signaling, Jun and Fos expression and subcellular localization were examined in genetic backgrounds that are either reduced or elevated with respect to JNK signaling. In bsk2 embryos, which are deficient in JNK activity, the amnioserosal cells show strong cytoplasmic localization of Jun and Fos. The cytoplasmic localization is clearly enhanced in bsk2/+ embryos, relative to wild-type, suggesting that nuclear versus cytoplasmic localization of Jun and Fos is particularly sensitive to reduction in JNK signaling levels. To test the effect of increasing JNK activity in the amnioserosa, puc mutant embryos were immunostained. In this background, JNK activity is upregulated, and both Jun and Fos were restricted to the nuclei of the amnioserosal cells throughout embryogenesis. This is the first report of nucleo-cytoplasmic regulation of Jun and Fos localization in Drosophila in response to JNK signaling. Jun and Fos nuclear localization as well as dpplacZ and puclacZ expression support the conclusion that JNK signaling occurs in the amnioserosa prior to dorsal closure. Reciprocally, the reduction of dpplacZ and puclacZ expression and the movement of Jun and Fos from the nucleus into the cytoplasm of amnioserosal cells are indicative of downregulation of JNK signaling in this tissue prior to and during dorsal closure (Reed, 2001).
Given the phenotypic similarities between hnt and JNK signaling mutants, the genetic interactions between hnt and the JNK pathway mutants and observations that JNK signaling is normally downregulated in the amnioserosa prior to dorsal closure, it was asked whether molecular confirmation for Hnt as a negative regulator of JNK signaling could be found (Reed, 2001).
Hnt is not required for Jun and Fos expression, since these proteins are present in the amnioserosa of hnt mutant embryos at levels roughly comparable to wild-type. Strikingly, in contrast to wild-type embryos, hnt mutant embryos (hnt308 and hntXO01) show persistent nuclear localization of Jun and Fos. These results are consistent with the postulated role of Hnt as a negative regulator of JNK signaling in the amnioserosa (Reed, 2001).
Persistent nuclear localization of Jun and Fos is seen, not only in the amnioserosa of hnt mutants, but also in the amnioserosa of puc mutants in which dorsal closure also fails. Thus hnt and puc mutants provide independent lines of evidence that downregulation of JNK signaling in the amnioserosa is essential for dorsal closure (Reed, 2001).
Formation or maintenance of focal complexes in the leading edge of the epidermis is disrupted by persistent JNK signaling in the amnioserosa. In wild-type embryos, phosphotyrosine and F-actin accumulate conspicuously along the dorsal-most leading edge cell membranes that abut the amnioserosa, representing focal complexes. Focal complexes fail to accumulate in leading edge cells of puc mutants. Similarly, in hnt mutants, phosphotyrosine and F-actin fail to accumulate at the dorsal-most membrane of the leading edge cells. Thus, Hnt function in the amnioserosa is necessary for the adjacent leading edge cells to assemble or maintain focal complexes at their dorsal-most membranes (Reed, 2001).
The failure of focal complex assembly in the leading edge cells of hnt and puc mutants is not a secondary consequence of the failure of JNK signaling in these cells. This conclusion derives from the fact that dpplacZ and puclacZ are expressed in the leading edge of wild-type, puc, and hnt mutants. Consistent with this result, the cells of the lateral epidermis undergo dorsal-ventral elongation in puc and hnt mutants, a process dependent on JNK-induced signals from the leading edge (Reed, 2001).
The simplest interpretation of these data is that assembly or maintenance of focal complexes in the leading edge occurs only if there is a boundary between high and low JNK signaling at the junction of the leading edge (high) and the amnioserosa (low). In hnt and puc mutants, since JNK signaling persists in the amnioserosa, such a high/low JNK activity boundary never forms, and therefore focal complexes are either not assembled or are not maintained at the dorsal membrane of the leading edge. In the absence of focal complexes, the leading edge is unable to move over the amnioserosa (Reed, 2001).
The hypothesis that focal complexes form only when there is a high/low JNK signaling boundary at the juxtaposition of the leading edge and the amnioserosa predicts that conversion of a high/high back to a high/low condition will lead to the restoration of focal complexes. Therefore JNK activity was downregulated in the amnioserosa of hnt mutants during the stages at which JNK signaling would abnormally persist. This was accomplished by expressing PUC or dominant-negative JNK using an amnioserosa-specific GAL4 driver. In hnt mutants (the high/high situation), focal complexes are absent from the leading edge, and the morphology of the leading edge cells is highly abnormal. Consistent with the hypothesis, when either Puk or dominant-negative JNK is expressed in the amnioserosa of hnt mutants, focal complexes are restored to the dorsal-most membrane of the leading edge, and the morphology of the leading edge is shifted toward wild-type (Reed, 2001).
In summary, these analyses show that focal complexes fail to accumulate in the leading edge when there is no JNK signaling boundary between the leading edge and the amnioserosa. The restoration of a high/low situation by the expression of either PUC or dominant-negative JNK in a hnt mutant results in the restoration of focal complexes in the leading edge (Reed, 2001).
Bases in 3' UTR - 163
Pebbled has 14 C2H2 type zinc fingers in five clusters. The first cluster of three domains begins at amino acid 247; the second cluster consists of two zinc finger domains begins at amino acid 513; the third cluster consists of three domains begins at amino acid 706; the fourth, a solitary domain, begins at amino acid 1056; the fifth, consisting of two domains begins at amino acid 1445, and the sixth cluster, consisting of three zinc finger domains, begins at amino acid 1620. There are multiple glutamine-rich domains, proline-rich domains, serine/threonine-rich domains and acidic/charged domains (Yip, 1997).
date revised: 13 September 2001
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