BIOLOGICAL OVERVIEW

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.

Downregulation of Jun kinase signaling in the amnioserosa is essential for dorsal closure of the Drosophila embryo

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 hnt^704a and hnt^XO01 successfully complete germ band retraction but do not hatch. Analyses of cuticle preparations have revealed that 60% of hnt^704a and 79% of hnt^XO01 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 dpp^lacZ and puc^lacZ 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 puc^lacZ and dpp^lacZ, 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 dpp^lacZ or puc^lacZ 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 puc^lacZ and dpp^lacZ 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 dpp^lacZ and puc^lacZ expression support the conclusion that JNK signaling occurs in the amnioserosa prior to dorsal closure. Reciprocally, the reduction of dpp^lacZ and puc^lacZ 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 hnt^XO01) 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 dpp^lacZ and puc^lacZ 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).

Hindsight mediates the role of Notch in suppressing Hedgehog signaling and cell proliferation

Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle (M/E switch). This study shows that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of cubitus interruptus. These studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation (Sun, 2007).

How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. These data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Sun, 2007).

Studies have shown that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature-cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed. The current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle (Sun, 2007).

The finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots, suggesting that an additional cell cycle also takes place. Further testing of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may not be the sole mediator of the Notch effect; for example, Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later, suggesting that Hnt function is also required for chorion-gene amplification (Sun, 2007).

The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells. Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots. The nuclear sizes of fu mutant cells were similar to those of the wild-type at the same developmental stage, and no fragmentation of the chromosomes was observed. Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus, Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of Ci-155 in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis. Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells (Sun, 2007).

Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells, but it is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt. Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Sun, 2007).

An interesting observation from these studies is that ttk clones have a phenotype similar to that of hnt clones. As in Notch regulation of Hnt, ttk is possibly downstream of Notch, but the current analysis of Notch mutants in stage-1 to stage-10 egg chambers showed no obvious change in Ttk expression. It was also found that Hnt has no role in regulating ttk expression. The findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis. The phenotypic similarity between hnt and ttk mutants suggests that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye. Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear (Sun, 2007).

Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt-deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process. Second, nurse-cell endoreplication does not require Hnt; no obvious defect was detected in hnt germline clones. The specific role of Hnt in follicle-cell-cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation (Sun, 2007).

Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on the cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway. Hh and Notch therefore act sequentially and positively during the SMW, whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis, but it is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling; mutation of the negative regulator of the Hh pathway, ptc, in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germline cells. These ptc mutant cells show no accumulation of Ci-155, consistent with the finding that Notch signaling suppresses ci transcription through Hnt. The ptc^S2 cells that were out of contact with germline cells remained in the mitotic cycle because they could not receive Dl signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state (Sun, 2007).

Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration. When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% of egg chambers showed defects in border-cell migration. Notch signaling, as well as ttk, has been reported to be required for border-cell migration. Hnt was found to be expressed in the border cells and depended on Notch signaling. The occasional hnt border-cell clones observed also showed defects in border-cell migration, so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells (Sun, 2007).

Transcription factor Pebbled/RREB1 regulates injury-induced axon degeneration

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 (Farley, 2018).

Neurons are connected over long distances by their axons, which can extend over more than a meter in humans. Maintenance of axon integrity is essential for sustained neural circuit function because axon breakage can block nervous system signal propagation. Axon loss is a hallmark of nervous system injuries, such as traumatic brain injury and spinal cord injury, is a unifying feature of neurodegenerative diseases, and is strongly correlated with functional loss in patients (Farley, 2018).

Wallerian degeneration (WD; axotomy) serves as a useful model to study basic aspects of axon biology, and to identify axon death signaling molecules. Severed axons, after a defined latent phase, undergo explosive fragmentation and are ultimately cleared by surrounding phagocytes. The discovery of the slow WD (WldS) mutant mouse, where severed distal axons survived for weeks after axotomy, radically changed our view of axonal biology. The observed long-term survival of distal severed axon fibers in Wld^S animals demonstrated that axon degeneration is a controlled process, and that under some conditions, axons could survive for weeks without a cell body. A growing number of studies across several species support the notion that the competence to undergo degeneration is likely a genetically programmed event: in the rock lobster, distal severed axons have been found to survive for a year after transection in vivo, and remain capable of evoked release at neuromuscular junctions; fragments of Aplysia axons can survive in vitro for extended periods of time without degeneration; and in Caenorhabditis elegans, most distal severed axons never degenerate. Despite these surprising observations, nothing is known about transcriptional mechanisms that regulate the competence of axons to degenerate (Farley, 2018).

In axons that do undergo WD, the execution of degeneration is driven by axon death signaling molecules. Drosophila dSarm (sterile α, ARM, and TIR domain protein) was the first endogenous molecule shown to actively promote axon death. Sarm1 functions in a conserved role in mammals, where it has been proposed to act as an NAD+ hydrolase that drives axonal degeneration through promoting metabolic catastrophe. Drosophila dSarm is similarly capable of NAD+ hydrolysis, but requires signaling downstream through the BTB/BACK domain molecule Axundead to execute axon death in vivo (Neukomm, 2017). The E3 ubiquitin ligase Highwire/Phr1 also modulates axon death signaling through a mechanism that appears to involve regulating levels of the NAD+ biosynthetic molecule dNmnat/Nmnat2. In both Drosophila and mammals, all neurons tested thus far have been strongly protected by loss-of-function mutations in dSarm/Sarm1, which suggests that axon death signaling molecules are engaged to drive destruction in a wide array of, or perhaps all, neuronal subtypes (Farley, 2018).

This study presents the identification and characterization of a role for Pebbled (Peb), a transcription factor, in axon degeneration. peb mutants show two predominant axon death-defective phenotypes: (i) severed distal axons are fully preserved morphologically, or (ii) the axon shaft breaks into large fragments (partially fragmented axons, PFAs) that fail to disintegrate further, lingering in the nervous system for weeks. The PFA phenotype in peb mutants is not observed in control or axon death mutants, and therefore defines a genetically accessible step in axon death signaling. Surprisingly, while PFAs form in all neurons, the ability of peb mutations to completely suppress axon degeneration was only observed in glutamatergic neurons, and not in cholinergic neurons, arguing that Peb functions to differentially modulate competence to undergo axon degeneration in distinct subsets of neurons (Farley, 2018).

Distal axons separated from their cell bodies can survive in a functionally competent state for days to weeks after axotomy in multiple species. It therefore seems plausible that some axons are programmed to degenerate while others are not. This study identified the transcription factor Pebbled (Peb)/RREB1 as an essential modulator of axon death in Drosophila. Through epistatic analysis, peb is placed upstream of dSarm or in a separate, parallel pathway, which ultimately converges on axon death. The simplest interpretation of the data is that Peb regulates axon death signaling in glutamatergic axons at the transcriptional level. Human RREB1 can rescue axon death phenotypes associated with loss of peb, arguing for strong conservation of the binding properties of Peb and hRREB1, and implying that RREB1 may play similar roles in axon biology in mammals (Farley, 2018).

Pebbled appears to identify a step in the axon death signaling cascade. Loss of Pebbled function resulted in the appearance of three axon phenotypes after injury: (i) full morphological preservation with a slightly delayed loss of mitochondria; (ii) the generation of PFAs that linger in the nervous system for weeks, but which also lose mitochondria after a short delay; or (iii) apparently normal axon death signaling and clearance. The cell biology of axon preservation in peb mutants is unique, and implies that peb mutants identify a genetically accessible step in axon death signaling. PFAs have not been observed in other axon death mutants (i.e., dsarm, hiw, or axed), as these mutants all fully block axon degeneration after axotomy. Understanding the nature of PFA production compared with normal axon degeneration is an important goal for future study. In the case of dsarm mutants, in addition to the axon shaft maintaining complete integrity, mitochondria also appeared well-preserved. That was not the case in peb mutants, where mitochondria degenerated after only a short delay, and preserved axons were severely depleted of mitochondria for the duration of their extended survival. Interestingly, the phenotype of individual peb mutant axons is consistent along the entirety of the axon shaft: an axon that generated PFAs in one portion, but was fully protected elsewhere, is never observed. Unraveling the molecular basis of this all-or-none type of phenotypic expression is of great interest for the future. Finally, while some PFAs are observed in control animals immediately after the initiation of axon fragmentation, they quickly undergo explosive degeneration and are cleared. From these data, it is concluded that Peb functions both at the initial phase of axon breakage into smaller fragments, and subsequently during the phase of explosive degeneration (Farley, 2018).

To date all known axon death signaling molecules -- dSarm, Hiw, and Axed -- have been proposed to function locally in the axon to drive destruction, and their neuroprotective effects extend to both glutamatergic and cholinergic neurons. Based on its expression in the nuclei of wing sensory neuron precursors and mature neurons, and the fact it is a C2H2 zinc finger transcription factor, it is proposed that Peb functions at the transcriptional level to help establish and maintain competence to undergo axon degeneration. While Peb appears to be expressed broadly in wing sensory neurons, surprisingly, the ability of peb mutants to fully block axon fragmentation is restricted to glutamatergic neurons. How Peb selectively protects glutamatergic axons is unclear, but could modify axonal phenotypes through the JNK signaling cascade. During embryogenesis, in AS peb mutants show increased levels of AP-1 transcriptional activity downstream of the JNK signaling cascade, which in turn inhibits cytoskeletal rearrangements that allow for cell migration. This study found a lack of evidence to support a role for JNK signaling in axon death, but some data support a neuroprotective role for this pathway by controlling baseline levels of Nmnat. Peb has also been shown to negatively regulate nervy, the Drosophila homolog of mammalian MTG8 proto-oncogene; however, this study observed no alterations in axon death when nervy was overexpressed in glutamatergic neurons (Farley, 2018).

The nuclear localization requirement of the carboxy terminal DNA binding zinc finger domains of Peb for rescue suggests that Peb is regulating injury-induced axon degeneration at the transcriptional level. Attempts were made to identify direct transcriptional targets of Peb by expressing a tagged version of Peb in the Drosophila embryonic cell line, GM2, and performing ChIP with antibodies specific to tagged Peb and subsequent deep sequencing (ChIP-seq). This approach successfully identified the one known target for Peb, the transcriptional regulator Nervy, whose overexpression did not block axon death, and several potential Peb targets. No evidence was found for direct binding of Peb to regions containing known axon death signaling genes (i.e., dsarm, axed, or hiw). This could indicate that Peb does not directly modulate axon death genes in vivo to exert its effects, although it remains unclear how similar Peb transcriptional activity in GM2 cells might be compared with neurons. Finally, it remains unclear why dsarm, but not axed mutations, modify peb mutant phenotypes, given that Axed signals downstream of dSarm in axon death. A deeper understanding of the molecular basis of axon degeneration, and the nature of PFAs, will likely be required to answer this question (Farley, 2018).

This analysis of peb mutant phenotypes reveals features of the cell biology of axon death. The discovery of PFAs in peb mutants implies that axon degeneration can be genetically dissected into distinct activation and execution phases, and it is proposed that PFAs represent activation, but failure to execute axon death. Either increased or decreased Peb levels could lead to the production of PFAs, arguing that fine-tuning of Peb levels is essential for appropriate execution of axon death. Furthermore, Peb modulation of PFA production is not limited to glutamatergic neurons, since PFAs were found in cholinergic neurons under both loss- and gain-of-function Peb conditions. Interestingly, Peb allows genetic separation of mitochondrial loss from axon degeneration. Mitochondrial degeneration was observed even in fully protected peb mutant axons, indicating that mitochondrial destruction must occur through a Peb-independent signaling pathway (Farley, 2018).

GENE STRUCTURE

Bases in 3' UTR - 163

PROTEIN STRUCTURE

Amino Acids - 1920

Structural Domains

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: 5 December 2023  

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