InteractiveFly: GeneBrief

pecanex: Biological Overview | References

BIOLOGICAL OVERVIEW

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. This study has established that pcx is a component of the N-signaling pathway. pcx is required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function results in enlargement of the ER that is not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppresses the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes is induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, it is speculated that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. These results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity (Yamakawa, 2012).

Cell-cell signaling mediated by the Notch (N) receptor is implicated in a wide variety of developmental processes in multicellular organisms, across phyla. In humans, N-signaling abnormalities cause diseases that include leukemia, other cancers, and pulmonary arterial hypertension. Drosophila N encodes a transmembrane receptor with 36 epidermal growth factor (EGF)- like repeats in its extracellular domain. During maturation of N, its extracellular domain is cleaved by Furin protease (S1 cleavage) in the Golgi. After reaching the cell surface, the binding of N to its transmembrane ligand, Delta or Serrate, leads to a second cleavage in the extracellular domain of N by Kuzbanian (Kuz)/ADAM10 or ADAM17 (S2 cleavage). This cleavage removes most of the N extracellular domain and produces a membrane-tethered form of the N intracellular domain (NEXT). Subsequently, NEXT is cleaved within its transmembrane domain by γ-secretase (S3 cleavage), which liberates the intracellular domain, termed NICD. NICD then translocates to the nucleus and regulates the transcription of downstream genes (Yamakawa, 2012).

N requires various post-translational modifications to its extracellular domain to be activated. For example, O-glycosylation of the N extracellular domain by O-fucosyltransferase 1 (O-fut1) and Fringe regulates the binding between N and its ligands. O-fut1 is also known to act as an N-specific chaperone in Drosophila. In addition, analysis of a Drosophila thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1-like (Ero1L), showed that disulfide-bond formation in the extracellular domain of N is indispensable for the activation of the N signal (Yamakawa, 2012).

Many roles played by N signaling in Drosophila development are crucial and have been studied extensively. Its best-known role during the early development of the central nervous system, is to prevent cells that neighbor a neuroblast from choosing the neuroblast fate, a phenomenon called 'lateral inhibition'. This is achieved when the neuroblast-fated cell activates N signaling in its neighbors; these cells become epidermoblasts. Thus, disruption of N signaling in Drosophila embryos results in the failure of lateral inhibition and the consequent hyperplasia of neuroblasts at the expense of epidermoblasts, which is referred to as the 'neurogenic" phenotype. Because most of the genes that encode N-signaling components are essential for lateral inhibition, these genes were first identified by the neurogenic phenotype resulting from their disruption (Yamakawa, 2012).

pecanex was originally identified as a mutant showing recessive female sterility (Perrimon, 1984). Thus, pcx homozygous or hemizygous embryos obtained from pcx heterozygous females survive until adulthood. However, embryos obtained from pcx homozygous females mated with pcx hemizygous males, which are fertile, show neuronal hyperplasia, i.e. the neurogenic phenotype, suggesting that the maternally supplied pcx function rescues this phenotype (LaBonne, 1985). Therefore, pcx is considered to be a maternal neurogenic gene. pcx encodes a multi-pass transmembrane protein consisting of 3433 amino acids that is highly conserved from Drosophila to humans (LaBonne, 1989). A rat homolog of pcx, pecanex1, is expressed in spermatocytes and probably functions in the testes (Geisinger, 2005). However, no molecular function of the pcx protein has been identified in any species. This study has established that pcx is an N-signaling component in Drosophila. Evidence is also provided that pcx might be involved in ER functioning (Yamakawa, 2012)

No motifs that might suggest pcx's biochemical function have been found in its amino acid sequence. Although pcx was previously suggested to be involved in N signaling, based on the neurogenic phenotype associated with its mutant in Drosophila, this possibility had not been explored. This study provides evidence that pcx is a component of the N-signaling pathway (Yamakawa, 2012).

In pcx^m/z embryos, the ER was abnormally enlarged. Various factors regulating the architecture of the ER have been identified. In Drosophila, Atlastin, a dynamin-like GTPase, is required for fusion of the ER membrane (Orso, 2009). Thus, the overexpression of Atlastin induces an enlarged ER (Orso, 2009). In addition, the peripheral ER shows two distinct structures: tubules and sheets. Several factors organizing the shape of the ER membrane into tubules or sheets have been identified (English, 2009). Therefore, pcx might contribute to the regulatory machinery that accomplishes the normal organization of the ER (Yamakawa, 2012).

In pcx^m/z embryos, the enlarged ER was observed predominantly in the region corresponding to the dorsal epidermis of wild-type embryos. Therefore, sensitivity to the absence of pcx function might differ among groups of cells. This distinct behavior could reflect differences in the cell-cycle phase or level of UPR activity (Yamakawa, 2012).

Although the results showed that the reduction of N signaling was not responsible for the enlargement of the ER in pcx^m/z embryos, the ectopic activation of N signaling by overexpression of NICD also suppressed this ER defect. It is speculated that the ectopic activation of N signaling might affect the progression of the cell-cycle or the level of UPR, which could in turn affect the regulation of the ER architecture. It has been shown that N signaling directly or indirectly affects the cell cycle. However, the biological significance and mechanisms of this phenomenon remain elusive (Yamakawa, 2012).

Induction of the UPR was found to suppress the ER enlargement in pcx^m/z embryos. The suppression of the ER enlargement by the expression of genes that induce the UPR coincided with the rescue of N signaling activity in these embryos. Therefore, the reduced N signaling in pcx^m/z embryos might be attributable to the enlargement of the ER. However, the possibility cannot be excluded that pcx is independently involved in the activation of N signaling and the regulation of the ER architecture. Nevertheless, the results suggest that some downstream events induced by the UPR compensate for the defect of N signaling associated with the absence of pcx function. It was found that overexpression of O-fut1, an N-specific chaperone, partially compensated for the loss of pcx function. Thus, a disruption of N signaling in the absence of pcx function might be partly due to the mis-folding of N, which is consistent with the hypothesis that pcx acts upstream of the activated forms of N and probably functions in signal-receiving cells (Yamakawa, 2012).

The UPR induces various downstream events, including the attenuation of protein synthesis, the enhancement of misfolded ER protein degradation, and the induction of genes encoding various chaperones. Therefore, in future experiments, it will be important to determine the specific defects that are compensated for by the UPR in the absence of pcx function (Yamakawa, 2012).

Molecular genetics of pecanex, a maternal-effect neurogenic locus of Drosophila melanogaster that potentially encodes a large transmembrane protein

In the absence of maternal expression of the pecanex gene, the embryo develops severe hyperneuralization similar to that characteristic of Notch mutant embryos. A previous molecular analysis of the chromosomal interval that encompasses pecanex was extended by using additional deficiencies to localize the locus on the molecular map. RNA blot analysis shows that the locus encodes a rare 9-kb transcript as well as minor transcripts of 3.7 and 2.3 kb. The temporal expression of these transcripts is appropriate for a neurogenic locus. Phenocopies of the mutant phenotype have been produced following microinjection of antisense RNA corresponding to a portion of the pecanex transcripts. Conceptual translation of a partial coding sequence compiled from cDNA and genomic clones indicates that the pecanex locus potentially encodes a large, membrane-spanning protein (LaBonne, 1989).

Search PubMed for articles about Drosophila Pecanex

English, A. R., Zurek, N. and Voeltz, G. K. (2009). Peripheral ER structure and function. Curr. Opin. Cell Biol. 21: 596-602. PubMed ID: 19447593

Geisinger, A., Alsheimer, M., Baier, A., Benavente, R. and Wettstein, R. (2005). The mammalian gene pecanex 1 is differentially expressed during spermatogenesis. Biochim. Biophys. Acta 1728(1-2): 34-43. PubMed ID: 15777640

LaBonne, S. G. and Mahowald, A. P. (1985), Partial rescue of embryos from two maternal-effect neurogenic mutants by transplantation of wild-type ooplasm. Dev. Biol. 110(1): 264-7. PubMed ID: 4007265

LaBonne, S. G., Sunitha, I. and Mahowald, A. P. (1989). Molecular genetics of pecanex, a maternal-effect neurogenic locus of Drosophila melanogaster that potentially encodes a large transmembrane protein. Dev. Biol. 136(1): 1-16. PubMed ID: 2478400

Orso, G., et al. (2009). Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin. Nature 460: 978-983. PubMed ID: 19633650

Perrimon, N., Engstrom, L. and Mahowald, A. P. (1984). Developmental genetics of the 2E-F region of the Drosophila X chromosome: a region rich in 'developmentally important' genes. Genetics 108(3): 559-72. PubMed ID: 6437900

Yamakawa, T., et al. (2012). Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila. Development 139(3): 558-67. PubMed ID: 22190636

date revised: 30 October 2012

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