Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila.

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. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was 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 resulted in enlargement of the ER that was 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 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate 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. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.

An unconventional myosin in Drosophila reverses the default handedness in visceral organs.

The internal organs of animals often have left-right asymmetry. Although the formation of the anterior-posterior and dorsal-ventral axes in Drosophila is well understood, left-right asymmetry has not been extensively studied. Here we find that the handedness of the embryonic gut and the adult gut and testes is reversed (not randomized) in viable and fertile homozygous Myo31DF mutants. Myo31DF encodes an unconventional myosin, Drosophila MyoIA (also referred to as MyoID in mammals; refs 3, 4), and is the first actin-based motor protein to be implicated in left-right patterning. We find that Myo31DF is required in the hindgut epithelium for normal embryonic handedness. Disruption of actin filaments in the hindgut epithelium randomizes the handedness of the embryonic gut, suggesting that Myo31DF function requires the actin cytoskeleton. Consistent with this, we find that Myo31DF colocalizes with the cytoskeleton. Overexpression of Myo61F, another myosin I (ref. 4), reverses the handedness of the embryonic gut, and its knockdown also causes a left-right patterning defect. These two unconventional myosin I proteins may have antagonistic functions in left-right patterning. We suggest that the actin cytoskeleton and myosin I proteins may be crucial for generating left-right asymmetry in invertebrates.

Three enhancer regions regulate gbx2 gene expression in the isthmic region during zebrafish development.

In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.

Gbx2 functions as a transcriptional repressor to regulate the specification and morphogenesis of the mid-hindbrain junction in a dosage- and stage-dependent manner.

The Gbx subfamily of homeodomain transcription factors is involved in the positioning of the isthmus, which patterns the midbrain and cerebellum in vertebrates. To uncover the details of Gbx functions, we first examined the dose dependency of its effects on brain formation in zebrafish and found that high-dose gbx2 mRNA injection affected the entire forebrain and midbrain, whereas low-dose mRNA specifically disrupted the isthmic folding at the midbrain-hindbrain boundary (MHB) but only weakly affected the expression of genes involved in MHB specification. Thus, isthmus morphogenesis, and not its early specification, is highly sensitive to gbx2. Transient induction of heat-inducible gbx2 using transgenic fish showed that MHB specification is most sensitive to gbx2 at the end of epiboly and further suggested that otx2 is the direct target gene. These together demonstrate that gbx2 regulates both specification and morphogenesis of the MHB/isthmus region. Deletion analyses showed that both the N- and C-terminal regions contribute to the suppressive activity of Gbx2 against the anterior brain and that the N-terminal core region, including the Eh1 and proline-rich sequences, is required for this Gbx2 activity. Comparison of the effects of activated and repressive forms with wild-type Gbx2 suggested that Gbx2 functions as a transcriptional repressor, which was further evidenced by a luciferase assay in which gbx2 repressed the MHB enhancer of fgf8a in mouse P19 cells.

Drosophila HOPS and AP-3 complex genes are required for a Deltex-regulated activation of notch in the endosomal trafficking pathway.

DSL ligands promote proteolysis of the Notch receptor, to release active Notch intracellular domain (N(ICD)). Conversely, the E3 ubiquitin ligase Deltex can activate ligand-independent Notch proteolysis and signaling. Here we show that Deltex effects require endocytic trafficking by HOPS and AP-3 complexes. Our data suggest that Deltex shunts Notch into an endocytic pathway with two possible endpoints. If Notch transits into the lysosome lumen, it is degraded. However, if HOPS and AP-3 deliver Notch to the limiting membrane of the lysosome, degradation of the Notch extracellular domain allows subsequent Presenilin-mediated release of N(ICD). This model accounts for positive and negative regulatory effects of Deltex in vivo. Indeed, we uncover HOPS/AP-3 contributions to Notch signaling during Drosophila midline formation and neurogenesis. We discuss ways in which these endocytic pathways may modulate ligand-dependent and -independent events, as a mechanism that can potentiate Notch signaling or dampen noise in the signaling network.

Head region of unconventional myosin I family members is responsible for the organ-specificity of their roles in left-right polarity in Drosophila.

In Drosophila, Myosin31DF (Myo31DF), encoding a Myosin ID protein, has crucial roles in left-right (LR) asymmetric development. Loss of Myo31DF function leads to laterality inversion for many organs, including the embryonic gut. Here, we found that Myo31DF was required before LR asymmetric morphogenesis in the hindgut, suggesting it functions in LR patterning instead of directly in hindgut morphological changes. Myosin61F (Myo61F) encodes another Myosin I, and Myo31DF or Myo61F overexpression reverses the laterality of different organs. Myo31DF and Myo61F have domains conserved in Myosin proteins, particularly in the proteins' head regions. We studied the roles of these domains in LR patterning using overexpression analysis. The Actin-binding and ATP-binding domains were essential for both proteins, but the IQ domains, binding sites for Myosin light chains, were required only by Myo31DF. Our results also suggest that the organ specificities of the Myo31DF and Myo61F activities depended on their head regions.

Polarized exocytosis and transcytosis of Notch during its apical localization in Drosophila epithelial cells.

Notch (N) and its ligands, Delta (Dl) and Serrate (Ser), are transmembrane proteins that mediate the cell-cell interactions necessary for many cell-fate decisions. In Drosophila, N is predominantly localized to the apical portion of epithelial cells, but the mechanisms and functions of this localization are unknown. Here, we found N, Dl, and Ser were mostly located in the region from the subapical complex (SAC) to the apical portion of the adherens junctions (AJs) in wing disc epithelium. N was delivered to the SAC/AJs in two phases. First, polarized exocytosis specifically delivered nascent N to the apical plasma membrane and AJs in an O-fut1-independent manner. Second, N at the plasma membrane was relocated to the SAC/AJs by Dynamin- and Rab5-dependent transcytosis; this step required the O-fut1 function. Disruption of the apical polarity by Drosophila E-cadherin (DEcad) knock down caused N and Dl localization to the SAC/AJs to fail. N, but not Dl, formed a specific complex with DEcad in vivo. Finally, our results suggest that juxtacrine signaling in epithelia generally depends on the apicobasally polarized structure of epithelial cells.

The O-fucosyltransferase O-fut1 is an extracellular component that is essential for the constitutive endocytic trafficking of Notch in Drosophila.

Notch is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell-fate decisions. Endocytic trafficking of Notch plays important roles in the activation and downregulation of this receptor. A Drosophila O-FucT-1 homolog, encoded by O-fut1, catalyzes the O-fucosylation of Notch, a modification essential for Notch signaling and ligand binding. It was recently proposed that O-fut1 acts as a chaperon for Notch in the endoplasmic reticulum and is required for Notch to exit the endoplasmic reticulum. Here, we report that O-fut1 has additional functions in the endocytic transportation of Notch. O-fut1 was indispensable for the constitutive transportation of Notch from the plasma membrane to the early endosome, which we show was independent of the O-fucosyltransferase activity of O-fut1. We also found that O-fut1 promoted the turnover of Notch, which consequently downregulated Notch signaling. O-fut1 formed a stable complex with the extracellular domain of Notch. In addition, O-fut1 protein added to conditioned medium and endocytosed was sufficient to rescue normal Notch transportation to the early endosome in O-fut1 knockdown cells. Thus, an extracellular interaction between Notch and O-fut1 is essential for the normal endocytic transportation of Notch. We propose that O-fut1 is the first example, except for ligands, of a molecule that is required extracellularly for receptor transportation by endocytosis.

gbx2 Homeobox gene is required for the maintenance of the isthmic region in the zebrafish embryonic brain.

We isolated cDNA clones for the zebrafish gbx2 gene, which is implicated in the establishment of the midbrain-hindbrain boundary (MHB) in other vertebrates. Spatially localized expression of gbx2 was observed at the MHB from 90% epiboly through to the hatching stage. Comparisons with the expression of otx2, wnt1, and krox20 showed that gbx2 is expressed in the anterior hindbrain. Ectopic expression of gbx2 by mRNA injection caused cyclopia or truncation of the fore- and midbrain and severely affected isthmic and cerebellar structures, while hindbrain formation was not significantly affected. At the molecular level, gbx2 suppressed the expression of otx2 in the fore/midbrain, six3 in the anterior forebrain, and MHB-specific genes such as eng2 and wnt1. In contrast, gbx2 did not down-regulate the expression of the hindbrain marker genes. Therefore, gbx2 specifically suppressed the formation of the entire fore/midbrain. Meanwhile, misexpression of otx2 suppressed the expression of gbx2 in the embryonic brain. Abrogation of gbx2 expression with an antisense morpholino oligonucleotide disrupted the midbrain/anterior hindbrain region, and these loss-of-function effects were rescued by activating the Gbx2 protein immediately after the end of gastrulation. Taken together, these results suggest that the zebrafish gbx2 gene is essential for the maintenance of MHB and/or the formation of the isthmic structure during somitogenesis, rather than for the MHB establishment during gastrulation. We also suggest that other factors, including gbx1, is required for the establishment of the MHB during gastrulation.