The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.
Summary
Background
Altered expression of apicobasal polarity factors is associated with cancer in vertebrates and tissue overgrowth in invertebrates, yet mechanisms by which these factors affect growth regulatory pathways are not well defined. We have tested the basis of an overgrowth phenotype driven by the Drosophila protein Crumbs (Crb), which nucleates an apical membrane complex that functionally interacts with the Par6/Par3/aPKC and Scrib/Dlg/Lgl apicobasal polarity complexes.
Results
We find that Crb-driven growth is dependent upon the Salvador-Warts-Hippo (SWH) pathway and its transcriptional effector Yorkie (Yki). Expression of the Crb intracellular domain elevates Yki activity and this correlates in tissues and cultured cells with loss of Expanded (Ex), an apically localized SWH component that inhibits Yki. Reciprocally, loss of crb elevates Ex levels, although this excess Ex does not concentrate to its normal location at apical junctions. The Ex-regulatory domain of Crb maps to the juxtamembrane FERM-domain binding motif (JM), a cytoskeletal interaction domain distinct from the PDZ-binding motif (PBM) through which Crb binds polarity factors. Expression of Crb-JM drives Yki activity and organ growth with little effect on tissue architecture, while reciprocally Crb-PBM produces tissue architectural defects without significant effect on Yki activity.
Conclusions
These studies identify Crb as a novel SWH regulator via JM-dependent effects on Ex levels and localization, and show that discrete domains within Crb may allow it to integrate junctional polarity signals with a conserved growth pathway.
Some pathogens are able to establish themselves within the host because they have evolved mechanisms to disrupt host innate immunity. For example, a number of pathogens secrete preformed effector proteins via type III secretion apparati that influence innate immune or apoptotic signaling pathways. One group of effector proteins that usurp innate immune signaling is the YopJ-like family of bacterial effector proteins, which includes AopP from Aeromonas salmonicida. Aeromonas species are known to cause gastrointestinal disease in humans, and are associated mainly with subcutaneous wound infections and septicemia in other metazoans, particularly fish. AopP has been reported to have inhibitory activity against the NF-κB pathway in cultured cells, although the pathological outcomes of AopP activity have not been examined. Here, we show that AopP has potent pro-apoptotic activity when expressed in cultured mammalian macrophage or epithelial cells, or when ectopically expressed in Drosophila melanogaster hemocytes or imaginal disk epithelial cells. Furthermore, apoptosis was significantly elevated upon concurrent AopP expression and TNF-α cellular stimulation. Together, our results demonstrate how the specificity of a YopJ-like protein toward signaling pathways directly governs cellular pathological outcome in disease.
The tracheal system of Drosophila melanogaster is an interconnected network of gas-filled epithelial tubes that develops during embryogenesis and functions as the main gas-exchange organ in the larva. Larval tracheal cells respond to hypoxia by activating a program of branching and growth driven by HIF-1α/sima-dependent expression of the breathless (btl) FGF receptor. By contrast, the ability of the developing embryonic tracheal system to respond to hypoxia and integrate hard-wired branching programs with sima-driven tracheal remodeling is not well understood. Here we show that embryonic tracheal cells utilize the conserved ubiquitin ligase dVHL to control the HIF-1 α/sima hypoxia response pathway, and identify two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes: a relatively hypoxia-resistant ‘early’ phase during which Sima activity conflicts with normal branching and stunts migration, and a relatively hypoxia-sensitive ‘late’ phase during which the tracheal system uses the dVHL/sima/btl pathway to drive increased branching and growth. Mutations in the archipelago (ago) gene, which antagonizes btl transcription, re-sensitize early embryos to hypoxia, indicating that their relative resistance can be reversed by elevating activity of the btl promoter. These findings reveal a second type of tracheal hypoxic response in which Sima activation conflicts with developmental tracheogenesis, and identify the dVHL and ago ubiquitin ligases as key determinants of hypoxia sensitivity in tracheal cells. The identification of an early stage of tracheal development that is vulnerable to hypoxia is an important addition to models of the invertebrate hypoxic response.
The archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila melanogaster and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. Here we show that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease.
Mammalian cancers depend on ‘multiple hits’, some of which promote growth, and some of which block apoptosis. We screened for mutations that require a synergistic block in apoptosis to promote tissue overgrowth, and identified myopic, the Drosophila homolog of the candidate tumor-suppressor and endosomal regulator His-domain-protein-tyrosine-phosphatase (HD-PTP). We find that Myopic regulates the Sav/Wts/Hpo (SWH) tumor suppressor pathway: Myopic PPxY motifs bind conserved residues in the WW domains of the transcriptional co-activator Yorkie and Myopic colocalizes with Yorkie at endosomes. Myopic controls Yorkie endosomal association and protein levels, ultimately influencing expression of some Yorkie target genes. However, the anti-apoptotic gene diap1 is not affected, which may explain the conditional nature of the myopic growth-phenotype. These data establish Myopic as a Yorkie-regulator and implicate Myopic-dependent association of Yorkie with endosomal compartments as a regulatory step in nuclear outputs of the SWH pathway.
The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls adult locomotion, axon projection, dendritic arborization, and memory through a largely undefined set of target RNAs. Here, we show a specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and focus on retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl) that is enriched in female neurons. Previous studies have revealed that this splicing event is regulated in females by N6-methyladenosine (m6A) modification by the Mettl3 complex. At a molecular level, Nab2 associates with Sxl pre-mRNA in neurons and limits Sxl m6A methylation at specific sites. In parallel, reducing expression of the Mettl3, Mettl3 complex components, or the m6A reader Ythdc1 rescues mutant phenotypes in Nab2 flies. Overall, these data identify Nab2 as an inhibitor of m6A methylation and imply significant overlap between Nab2 and Mettl3 regulated RNAs in neuronal tissue.
Drosophila melanogaster is an excellent model organism to study neurodegeneration. Assessing evident neurodegeneration within the fly brain involves the laborious preparation of thin-sectioned H&E-stained heads to visualize brain vacuole degeneration. Here, we present an advanced microscopy-based protocol, without the need for sectioning, to detect vacuole degeneration within whole fly brains by applying commonly used stains to reveal the brain parenchyma. This approach preserves the whole-brain architecture and enables rapid, reproducible, and quantitative analyses of vacuole-like degeneration associated with specific brain regions. For complete details on the use and execution of this protocol, please refer to Behnke et al. (2021).
The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA-binding protein, is mutated in an inherited form of autosomal recessive, nonsyndromic intellectual disability. To gain insight into neurological functions of ZC3H14, we previously developed a Drosophila melanogaster model of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model revealed that Nab2 controls final patterns of neuron projection within fully developed adult brains, but the role of Nab2 during development of the Drosophila brain is not known. Here, we identify roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies, which support olfactory learning and memory, and regulating abundance of a small fraction of the total brain proteome. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule-binding protein Futsch, the neuronal Ig-family transmembrane protein turtle, the glial:neuron adhesion protein contactin, the Rac GTPaseactivating protein tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls the abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNAbinding proteins in neurodevelopment.
RNA-binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RNA-binding protein is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1- interacting RNA-binding protein Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we present an analysis of a brain proteomic dataset that indicates that multiple planar cell polarity proteins are affected by Nab2 loss, and couple this with genetic data that demonstrate that Nab2 has a previously unappreciated role in restricting the growth and branching of dendrites that elaborate from larval body-wall sensory neurons. Further analysis confirms that Nab2 loss sensitizes sensory dendrites to the genetic dose of planar cell polarity components and that Nab2-planar cell polarity genetic interactions are also observed during Nab2-dependent control of axon projection in the central nervous system mushroom bodies. Collectively, these data identify the conserved Nab2 RNA-binding protein as a likely component of post-transcriptional mechanisms that limit dendrite growth and branching in Drosophila sensory neurons and genetically link this role to the planar cell polarity pathway. Given that mammalian ZC3H14 localizes to dendritic spines and controls spine density in hippocampal neurons, these Nab2-planar cell polarity genetic data may highlight a conserved path through which Nab2/ZC3H14 loss affects morphogenesis of both axons and dendrites in diverse species.