17 research outputs found
Loss of the spectraplakin short stop activates the DLK injury response pathway in drosophila
The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in Drosophila) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in Caenorhabditis elegans, a calcium-dependent mechanism, can activate DLK. Here we describe a new mechanism that activates DLK in Drosophila: loss of the spectraplakin short stop (shot). In a genetic screen for mutants with defective neuromuscular junction development, we identify a hypomorphic allele of shot that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. We demonstrate that both phenotypes are the result of overactivation of the DLK signaling pathway. We further show that, unlike mutations in the PHR ligase Highwire, loss of function of shot activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons
Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II
Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia
Homeotic Genes Autonomously Specify the Anteroposterior Subdivision of the Drosophila Dorsal Vessel into Aorta and Heart
AbstractThe embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Here, we demonstrate that homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. We provide evidence that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. We also show that Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart
A genetic screen for regulators of muscle morphogenesis in Drosophila
The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developing Drosophila body wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendon cells. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, including back seat driver, kon-tiki, thisbe, and tumbleweed, arguing our screen had the depth and precision to uncover myogenic genes. We also identified new alleles of spalt-major, barren, and patched that presumably disrupt independent muscle morphogenesis pathways. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be mapped and characterized. Our screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that regulate skeletal muscle topology
Rapid generation of hypomorphic mutations
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels
Epsin potentiates Notchpathway activity in Drosophilaand C. elegans
pathway are known to modulate the activity of different
signaling pathways. Epsins promote endocytosis and are
postulated to target specific proteins for regulated
endocytosis. Here, we present a functional link between the
Notch pathway and epsins. We identify the Drosophila
ortholog of epsin, liquid facets (lqf), as an inhibitor of
cardioblast development in a genetic screen for mutants
that affect heart development. We find that lqf inhibits
cardioblast development and promotes the development of
fusion-competent myoblasts, suggesting a model in which
lqf acts on or in fusion-competent myoblasts to prevent
their acquisition of the cardioblast fate. lqf and Notch
exhibit essentially identical heart phenotypes, and lqf
genetically interacts with the Notch pathway during
multiple Notch-dependent events in Drosophila. We
extended the link between the Notch pathway and epsin
function to C. elegans, where the C. elegans lqf ortholog acts
in the signaling cell to promote the glp-1/Notch pathway
activity during germline development. Our results suggest
that epsins play a specific, evolutionarily conserved role to
promote Notch signaling during animal development and
support the idea that they do so by targeting ligands of the
Notch pathway for endocytosis.ye
dbx mediates neuronal specification and differentiation through cross-repressive, lineage-specific interactions with eve and hb9
Individual neurons adopt and maintain defined morphological and
physiological phenotypes as a result of the expression of specific
combinations of transcription factors. In particular, homeodomain-containing
transcription factors play key roles in determining neuronal subtype identity
in flies and vertebrates. dbx belongs to the highly divergent H2.0
family of homeobox genes. In vertebrates, Dbx1 and Dbx2
promote the development of a subset of interneurons, some of which help
mediate left-right coordination of locomotor activity. Here, we identify and
show that the single Drosophila ortholog of Dbx1/2
contributes to the development of specific subsets of interneurons via
cross-repressive, lineage-specific interactions with the motoneuron-promoting
factors eve and hb9 (exex). dbx is
expressed primarily in interneurons of the embryonic, larval and adult central
nervous system, and these interneurons tend to extend short axons and be
GABAergic. Interestingly, many Dbx+ interneurons share a sibling
relationship with Eve+ or Hb9+ motoneurons. The
non-overlapping expression of dbx and eve, or dbx
and hb9, within pairs of sibling neurons is initially established as
a result of Notch/Numb-mediated asymmetric divisions. Cross-repressive
interactions between dbx and eve, and dbx and
hb9, then help maintain the distinct expression profiles of these
genes in their respective pairs of sibling neurons. Strict maintenance of the
mutually exclusive expression of dbx relative to that of eve
and hb9 in sibling neurons is crucial for proper neuronal
specification, as misexpression of dbx in motoneurons dramatically
hinders motor axon outgrowth