Inadequate sensitivity of existing XBP1 stress sensing systems can be overcome by improving the efficiency of unconventional splicing of xbp1 mRNA. Recent reports regarding the cellular localization of XBP1/HAC1 mRNA during its splicing allowed us to construct a highly sensitive HG stress indicator (Fig. ) that can visualize the activation of IRE1/XBP1 pathway at the third instar larval stage during normal Drosophila development (Figs. , , , and ). Several types of cells in the organs where we detected IRE1/XBP1 activity are known for having high secretory capacity.
In the larval brain, we found significant IRE1/XBP1 activity in glial cells (Fig. ). While glia had been originally thought to function as the structural support cells in the nervous system, it has been revealed that they play several important roles in the development and homeostasis of the nervous system. In Drosophila
, glial cells are classified into three classes (surface-, cortex-, and neuropil-associated glia), each of which is subdivided further morphologically (Stork et al. 2010
). Whether IRE1/XBP1 active glia is restricted to only a subtype of those glia, or more broadly, is currently under investigation.
In mammals, oligodendrocytes in the central nerve system and Schwann cells in the peripheral nerve system myelinate axons by producing a large amount of myelin membrane proteins, cholesterol, and membrane lipids through the secretory pathway. Recent reports suggested that ER stress in myelinating cells is important in the pathogenesis of various disorders of myelin (Pennuto et al. 2008
; Lin and Popko 2009
). Neuropil glia and peripheral glia in Drosophila
are the counterparts of oligodendrocytes and Schwann cells, respectively. Therefore, these cells are the candidates that show constitutive IRE1/XBP1 activity. Although Drosophila
glia do not generate myelin sheaths, they form multi-layered membrane sheaths around neurons that are morphologically similar to the myelin sheaths in mammals (Freeman and Doherty 2005
). Thus, it is possible that the IRE1/XBP1 active glia protect neurons from their deterioration through this ensheathment, thereby contributing to brain homeostasis. Further studies are expected to inform us of the pathological significance of IRE1/XBP1 functions in human glia.
As shown in Fig. , IRE1/XBP1 pathway does not appear to be active in neuron. However, we do not exclude the possibility of neuronal IRE1/XBP1 activation in the brain. In fact, slight neuronal IRE1/XBP1 activity was occasionally observed in the ventral nerve cord during our repeated experiments. In this study, we conclude that in the third instar larval brain, the IRE1/XBP1 pathway is predominantly activated in glia while the activation is not detectable in neurons.
The importance of IRE1/XBP1 activity in the gut has already been studied in Caenorhabditis elegans
and mammals (Shen et al. 2001
; Kaser et al. 2008
; Richardson et al. 2010
). We identified intra-tissue distribution of IRE1/XBP1 activity in the proventriculus region of the gut (Fig. ). In the larval midgut and hindgut, we observed an irregular distribution of IRE1/XBP1 active cells. These were not entero-endocrine cells, as they did not colocalize with anti-Prospero antibody that marks those cells (data not shown). Secretory intestinal cells in the midgut other than entero-endocrine cells (Casali and Batlle 2009
) including the intestinal stem cells are possible candidates for these IRE1/XBP1 active cells.
IRE1/XBP1 activity in the fly Malpighian tubules (analogous to the kidney in mammals) was also unexpected. The activity was detected throughout the organ, but not all of the cells were IRE1/XBP1 active (Fig. ). Although the Malpighian tubules are attached at the junction of the midgut and the hindgut, they are morphologically and functionally independent from both of them. Identification of the IRE1/XBP1 active cells in the gut and the Malpighian tubules might reflect a shared physiological function of both organs. One possible shared function may be the selective uptake of the essential molecules, including several metal ions, from the contents passing through those organs. IRE1/XBP1 pathway might regulate the function of some transporter channels in these organs. Drosophila
Malpighian tubules are expected to be one of the models for the mammalian diabetic kidney diseases that are associated with UPR activation (Cunard and Sharma 2011
In this study, we also identified IRE1/XBP1 activity in the trachea (Fig. ). Previous reports lead us to point out its relevance to glial IRE1/XBP1 activity (Pereanu et al. 2007
; Tsarouhas et al. 2007
). One of them showed that tracheal development in Drosophila
brain was controlled by signals from glia (Pereanu et al. 2007
). According to the report, the branches of cerebral trachea grow around the neuropile. If IRE1/XBP1 active glia were neuropile-associated glia, assessing IRE1/XBP1 activity at neuropile-associated glia is likely to allow us to reveal the shared physiological function of IRE1/XBP1 pathway between brain and trachea. The other report, using embryonic trachea, indicated that the proper combination of secretory activity and endocytotic activity was important for the maturation of trachea as an airway. In tracheal maturation, Sar1, one of the core COPII proteins, was required for the secretion of protein, the luminal matrix assembly, and the following expansion of tube diameter to avoid the clogging of protein, while Rab5, the small GTPase that regulates the early stage of endocytosis, was required for the clearance of deposited materials in the lumen (Tsarouhas et al. 2007
). It can be predicted that, even in larval trachea, IRE1/XBP1 pathway plays a crucial role in tracheal maturation by supplying the properly folded proteins to the transport machinery. In that case, in view of second instar larval lethality of xbp1
−/− hypomorph mutant, we could also hypothesize that the tracheal maturation/maintenance is still important for larval lethality, in addition to its importance for the embryonic development.
IRE1/XBP1 activity in the salivary gland has already been reported in a previous study (Souid et al. 2007
). The salivary gland is commonly used for the determination of the subcellular localization of the protein in Drosophila
cells due to its morphological features. Figure clearly indicated the nuclear localization of HG indicator, XBP1-EGFP molecule. In addition, we observed weak IRE1/XBP1 activity in the fat body that was attached to the salivary gland (Fig. ). Generally, the Drosophila
fat body, which is equivalent to mammalian adipose tissue, functions as the organ for energy/lipid storage and is distributed throughout the larval body.
In addition to the larval tissues, we analyzed IRE1/XBP1 activity in the adult male reproductive organs. Though the previous RT-PCR study by Souid et al. (2007
) suggested the activity in the testis, the areas we detected IRE1/XBP1 activity were the accessory glands and a limited area of the testis close to the testicular duct (Fig. ). In the accessory gland, seminal fluid containing several hormones, which facilitate reproductive traits such as sperm transfer, sperm storage, female receptivity, ovulation, and oogenesis, are produced and secreted (Wolfner 1997
; Chapman 2001
). There are two morphologically distinct secretory cell types in Drosophila
accessory gland. Ninety-six percent of the secretory cells are categorized as main cells and the others are secondary cells (Kalb et al. 1993
). Based on the intra-tissue distribution of IRE1/XBP1 active cells in the accessory gland, the active cells are likely to be main cells. Since each of these cell types expresses a unique set of genes, the confirmation of IRE1/XBP1 active cell type is expected to allow us to narrow down the proteins related to IRE1/XBP1 activity. IRE1/XBP1 pathway is likely to function, to some extent, in maintaining proper fertility.
On the other hand, we considered a possibility that the EGFP signal we detected in each organ might not necessarily reflect the unconventional splicing of xbp1-EGFP mRNA. Higher concentrations of the spliced xbp1-EGFP mRNA and resulting XBP1-EGFP in the cells induced by the Gal4/UAS system might cause the artifactual EGFP signal. We excluded the possibility that the EGFP signal in this study was detected independently of the unconventional splicing, based on our results in this study and the following reasoning.
There are two possible molecular mechanisms that cause the artifactual EGFP signal which is not derived from the unconventional splicing of xbp1-EGFP mRNA. One is the generation of EGFP or abnormal EGFP fusion proteins, resulting from translation initiation at the start codon of the EGFP coding sequence or at ATG codons coding Met residues in XBP1(s), respectively. The other is the proteolytic digestion of XBP1-EGFP fusion protein at the junction of XBP1 and EGFP portions. Both of these are prone to happen upon overexpression of fusion proteins in cells. In particular, the proteolytic digestion is often observed in the overexpression of GST fusion protein in Escherichia coli.
In our system, there is no nuclear localization signal (NLS) on the EGFP molecule. In contrast, xbp1 gene carries a NLS coding sequence located upstream of the unconventional splice site. There are a total of 11 ATG codons that code the Met residues of XBP1(s) molecule. Eight of the ATG codons are located downstream of NLS coding sequence. Therefore, due to the lack of NLS, both EGFP and the EGFP fusion proteins using these eight ATG codons as start codons should diffuse all over the cell upon their synthesis, if they were generated. As shown in Fig. , EGFP signal was detected exclusively in the nucleus in the salivary gland, which is often used for the analysis of the cellular localization of the proteins in Drosophila. Hence, it is not reasonable to conclude that either EGFP or the possible eight EGFP fusion proteins above were expressed in the cells. Only the EGFP fusion proteins that use the other three ATG codons that are upstream of the NLS as start codons should be synthesized upon unconventional splicing and localized in the nucleus. The estimated molecular weights of those fusion proteins are 73.7, 74.3, and 80.0 kDa, respectively. As shown in lane 4 (also lane 3) of Fig. , only a significant single band that represented the intact XBP1-EGFP (80.0 kDa) was detected in the S2 cell extract, in which XBP1-EGFP was overexpressed through the Gal4/UAS system. Therefore, we concluded that the EGFP fusion protein synthesized in this study was the intact XBP1-EGFP.
Additionally, there are several ATG codons that are located upstream of the unconventional splice site and are in frame with EGFP coding sequence on unspliced xbp1 mRNA. However, inside the 23 bp of unconventional-spliced fragment, there is a TGA stop codon that is also in frame with EGFP coding sequence. Even if the translation initiated from these start codons, the synthesis of these products should be terminated at this TGA stop codon before the ribosome would reach the EGFP coding region.
Regarding the proteolytic digestion of XBP1-EGFP fusion protein, the resulting EGFP should diffuse all over the cell upon its synthesis due to the lack of NLS. Therefore, the possibility of the proteolytic digestion is also excluded based on the same reasoning as above. Taken together, we concluded that the detected EGFP signal in this study exclusively reflected the occurrence of unconventional splicing of xbp1-EGFP mRNA.
In summary, we improved the sensitivity of the XBP1 stress sensing system and newly identified several organs where IRE1/XBP1 pathway is constitutively activated under normal physiological conditions. In particular, in the larval brain, significant glial specific activation was detected. Our improved system is expected to provide us with a number of clues to reveal the molecular mechanisms underlying the normal development and homeostasis controlled by IRE1/XBP1 pathway.