The expression of Traf1 during embryogenesis is highly dynamic and complex. Expression is observed in pre-blastoderm embryos indicative of a maternal complement that disappears after blastoderm formation. In these early embryos and throughout gastrulation, expression is most prominent in vitellophages. In contrast to the expression of murine TRAF4 (Masson, 1998), Traf1 transcripts are not predominant in neural tissues. However, they are detected in neural precursors -- the delaminating neuroblasts of the central, peripheral and stomatogastric nervous system of the embryo. Traf1 transcripts accumulate apically in embryonic neuroblasts already before the actual delamination process but quickly disappear thereafter and are no longer observed in ganglion mother cells or neurons. They are seen in a selection of presumptive sensory organ precursor cells in imaginal discs and a few neurons of the larval thoracic ganglion. The most consistent expression of Traf1 is observed in the mesoderm throughout development. Already before invagination, the presumptive mesoderm accumulates Traf1 mRNA. Expression can be followed throughout embryogenesis and is also detected in the adepithelial cells of wing imaginal discs, the presumptive imaginal muscle precursors. In contrast to other tissues, where Traf1 expression is rather transient, expression in the mesoderm persists throughout development except for terminally differentiated cells. In summary, Traf1 accumulates in mesodermal cells and neural precursors and is correlated with the onset of morphogenetic and cellular movements. It is largely absent from terminally differentiated cells (Preiss, 2001).
The larval brain accumulates Traf1 mRNA in the inner proliferation center of the optic lobe and in several single neuroblasts in the thoracic ganglion. In addition, the ring gland stains quite strongly. In the eye imaginal disc, Traf1 accumulation is seen behind the morphogenetic furrow in single cells or small groups of cells within the differentiating ommatidia. These cells may correspond to a subset of photoreceptor cells. In the antennal disc, mRNA staining is discernable in the center of the third and in a ring within the second segment. In the leg discs, single cells or small cell groups stain in those regions, from where proneural clusters for future mechano-sensory organs arise. In the prothoracic leg disc, the large cluster of the presumptive large chordotonal organ is most conspicuous. Wing discs stain most strongly in the adepithelial cells on the thoracic part that correspond to imaginal mesodermal precursor cells. Furthermore, staining is apparent on either side of the dorso-ventral boundary. It is stronger in the anterior compartment, where proneural clusters for mechano- and chemo-sensory organs of the wing margin arise. Also in the thoracic region, areas of presumptive proneural clusters stain (Preiss, 2001).
Traf1ex1 mutant failed to develop into the pupal stage. In order to understand the cause of the lethality, the internal organ structures of Traf1ex1 larvae were examined. The brain-ventral ganglia complex of the mutant larva showed no apparent defects compared to the wild-type control. However, interestingly, the mutant larva contained small-sized imaginal discs, especially the eye discs, in comparison to the wild-type discs. To examine the photosensory neuron projections in the brain hemisphere, eye-brain complexes were stained with monoclonal antibody 22C10, a well-characterized probe for sensory neurons in Drosophila. Axons from wild-type photoreceptors fan out evenly upon leaving the optic stalk and form a smooth neuronal array in the lamina. On the other hand, the photoreceptor axons from Traf1ex1 mutant form few axonal bundles and fail to defasciculate in the brain hemisphere. These findings suggest that Traf1 is indispensable for the development of imaginal eye discs and the formation of a correct photosensory neuronal array in the brain hemisphere (Cha, 2003).
During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC 'buds' off. This compensatory mechanism has been referred to as 'telophase rescue'. The Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (TRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. TRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires TRAF1. These data suggest that TRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway (Wang, 2006).
In telophase NBs, segregation of cell fate determinants, such as Pros, into future GMCs, is critical for their proper development. Telophase rescue appears to be one of the safeguard mechanisms that acts to ensure that GMCs inherit the cell fate determinants and adopt the correct cell identity when the mechanisms, which normally operate during NB divisions, fail (e.g., in insc mutant). Telophase rescue is a phenomenon for which the underlying mechanism involved remains largely unknown. The current data demonstrate that TRAF1 and Egr are two members of the Insc-independent telophase rescue pathway specific for Mira/Pros (Wang, 2006).
Although it is apically enriched in mitotic NBs and can directly interact with Baz in vitro, TRAF1 does not seem to be involved with the functions normally associated with the apical complex proteins. One distinct feature of TRAF1 differs from the other known apical proteins is its localization pattern; it is cytoplasmic in interphase and the apical crescent is prominent only at metaphase. In contrast, proteins of the apical complex are largely undetectable during interphase and form distinct apical crescents, starting from late interphase or early prophase. The protein localization difference between TRAF1 and other apical proteins suggests that TRAF1 and apical proteins are not always colocalized during mitosis. If TRAF1 is a bona fide member of the apical complex, the localization defects of other apical proteins are expected to be observed in TRAF1 mutant, as well as mislocalization of basal proteins, which was not detect. In addition, no spindle orientation or geometry defects were observed in the absence of TRAF1. Based on these observations, it is concluded that TRAF1 is not involved with the functions normally associated with the apical complex proteins (Wang, 2006).
The in vitro GST fusion protein pull-down assay suggests that TRAF1 may physically bind to Baz. This result is consistent with genetic data, indicating that TRAF1 acts downstream of baz and that its apical localization requires baz. These observations are consistent with the view that TRAF1 is recruited to the apical cortex by apical Baz in mitotic NBs. Baz, even at very low levels, can recruit TRAF1 to the apical cortex of the mitotic NBs. For example, in insc mutant NBs, TRAF1 remains apical probably owing to the low levels of Baz that remain localized to the apical cortex. This speculation is supported by Mira/Pros telophase rescue data, which clearly demonstrate that the telophase rescue seen in insc mutant NBs is severely damaged in baz mutant, suggesting that the Baz function required for Mira/Pros (and Pon/Numb) telophase rescue is intact in insc mutant NBs (Wang, 2006).
It has been shown that Pins/Gαi asymmetric cortical localization can be induced at metaphase by the combination of astral microtubules, kinesin Khc-73 and Dlg in the absence of Insc; this coincides with the observation that TRAF1 also forms tight crescent only at metaphase in both WT and insc mutant NBs. Does TRAF1 apical crescent formation also require the functions of astral microtubules, kinesin Khc-73 and Dlg? The data do not favor this hypothesis based on the following observations. (1) In TE35BC-3, a small deficiency uncovering sna family genes insc is not expressed but Pins and Gαi are asymmetrically localized, indicating that the astral microtubules, kinesin Khc-73 and Dlg pathway remain functional. TRAF1 is delocalized and is uniformly cortical in this deficiency line. (2) Similarly, in egr insc NBs, TRAF1 is cytoplasmic whereas the functions of astral microtubules, kinesin Khc-73 and Dlg are intact. (3) In egr NBs TRAF1 is cytoplasmic, whereas the apical complex is normal and astral microtubules, kinesin Khc-73 and Dlg are present. (4) TRAF1 apical localization remains unchanged in dlg mutant NBs. Based on these observations, it is concluded that TRAF1 apical localization is unlikely to share similar mechanism with Pins and Gαi and is likely to be independent of astral microtubules, kinesin Khc-73 and Dlg. TRAF1 apical localization appears to specifically require Egr and Baz (Wang, 2006).
In TRAF1 insc double-mutant embryos, the complete segregation of Mir/Pros into future GMCs occurs only in about 12% of the total population, and in the remaining NBs, only a fraction of Mira/Pros segregate into future GMCs as indicated by the Mira 'tail' extending into the future NBs at telophase. As it is difficult to address the global effect of this partial segregation of Mira/Pros on GMC specification in TRAF1 insc double mutant, focus was place on a well-defined GMC, GMC4-2a in NB4-2 lineage, to evaluate this issue. It is assumed that as long as the RP2 neuron (progeny of GMC4-2a, Even-skipped (Eve)-positive) was identified in a particular hemisegment, the GMC cell fate of GMC4-2a in that hemisegment should have been correctly specified. In insc mutants, almost all hemisegments contain RP2s, indicating that GMC4-2a has adopted the correct GMC cell fate in 99% of the total hemisegments. When TRAF1 insc double-mutant embryos were stained with anti-Eve, it was found the frequency of loss of Eve-positive RP2 neuron increased (to 8%) in late embryos, suggesting that about 8% of the GMCs in TRAF1 insc double mutant did not inherit sufficient Pros to specify the GMC fate in these embryos. The relatively low frequency (8%) of mis-specification of GMCs suggests that the threshold amount of Pros protein needed is sufficiently low such that just a partial inheritance of Pros, even when telophase rescue is compromised, is sufficient for most GMCs to be correctly specified (Wang, 2006).
Although Mira/Pros and Pon/Numb share similar basal localization patterns in insc NBs, further removal of either TRAF1 or Egr compromised telophase rescue only for Mira/Pros, but not for Pon/Numb. This difference between Mira/Pros and Pon/Numb indicates that the detailed mechanisms of basal localization and segregation of Mira/Pros differ from those of Pon/Numb, which is consistent with the observations that the dynamics of Mira/Pros and Pon/Numb localization early in mitosis are different and the basal localization for Mira/Pros and Pon/Numb requires different regions of the Insc coding sequence (Wang, 2006).
Dlg/Lgl/Scrib are required for correct basal localization of Mira/Pros and Pon/Numb in mitotic NBs. Dlg has been shown to be involved in the Mira telophase rescue. In dlg insc double-mutant NBs, not only was spindle geometry symmetric but Mira telophase rescue was also affected. It would be interesting to know if Dlg belongs to the same pathway as TRAF1 and Egr and if Dlg is also involved in Pon/Numb telophase rescue (Wang, 2006).
Two other members of the TRAF family have also been identified in Drosophila: DTRAF2 (DTRAF6) and DTRAF3. In contrast to the specific and strong expression of TRAF1 in the embryonic NBs, only low levels of ubiquitous signals similar to the control background were seen in the NBs with DTRAF2 and DTRAF3 probes. It is likely that DTRAF2 and DTRAF3 are not expressed in NBs and do not play an important role in Mira/Pros telophase rescue pathway as the Mira/Pros telophase rescue is dramatically compromised in TRAF1 insc and egr insc NBs (Wang, 2006).
In mammals, the TNF pathway works as a typical receptor-mediated signal transduction pathway. TNFR is a key player in transducing external signal to the cytoplasm. In the Drosophila compound eyes, ectopic Egr, Wgn and TRAF1 seem to work in a similar receptor-mediated signal pathway to induce apoptosis through the activation of the JNK pathway. Does the same Egr, Wgn and TRAF1 receptor-mediated signal pathway play a role in Mira/Pros telophase rescue? If it does, the coexpression of Egr, Wgn and TRAF1 might be expected to be seen in dividing NBs, along with the potential interaction between TRAF1 and the cytoplasmic domain of Wgn. Three observations argue against this hypothesis: (1) wgn is not expressed in embryonic NBs but in the mesoderm. (2) The domain analysis suggests that the Drosophila Wgn cytoplasmic domain is unique with no sequence homology to any mammalian TNFR family members and has neither a TRAF-binding domain nor a death domain, which is required for the interaction between TNFR and TRAF in mammals. (3) More informatively, Wgn knockdown by a UAS head-to-head inverted repeat construct of wgn (UAS-wgn-IR) driven by a strong maternal driver, mata-gal4 V32A, in WT embryos did not affect TRAF1 apical localization. These observations are consistent with the view that the receptor Wgn may not be involved in Mira/Pros telophase rescue or is redundant in this pathway. If this is the case, then how do TRAF1 and Egr function in Mira/Pros telophase rescue? It has been reported that TRAFs associate with numerous receptors other than the TNFR superfamily in mammals. It is speculated that Egr and TRAF1 may adopt an alternative receptor in NBs for Mira/Pros telophase rescue. However, until an anti-Wgn antibody and wgn mutant alleles are available, the possibility that Wgn is involved in Mira/Pros telophase rescue cannot be ruled out (Wang, 2006).
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date revised: 10 April 2008
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