αTubulin 67C and Ncd Are Essential for Establishing a Cortical Microtubular Network and Formation of the Bicoid mRNA Gradient in Drosophila

Abstract

The Bicoid (Bcd) protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localized at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfil the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. We report hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (i) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (ii) long MTs up to 50 µm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the bcd mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. Our data strongly corroborate the ARTS model and explain the occurrence of the bcd mRNA gradient.

DrosophilaTau Negatively Regulates Translation and Olfactory Long-Term Memory, But Facilitates Footshock Habituation and Cytoskeletal Homeostasis

“…Proteins were transferred to PVDF membrane and probed with the following monoclonal antibodies: N2 7A1 Armadillo [Developmental Studies Hybridoma Bank (DSHB)] at 1:200, E7 ␤-tubulin 97EF (DSHB) at 1:250, ADL84.12 Lamin Dm0 (DSHB) at 1:100, acetyl-␣-tubulin (Cell Signaling Technology) at 1:2000, FLAGM2 (Sigma-Aldrich) at 1:1000, and 8C3 syntaxin (DSHB) at 1:3000. Anti-␣Tub84BϩD guinea pig polyclonal antibody (1:300) was a gift from Stefan Baumgartner (Lund University, Sweden) (Fahmy et al, 2014), rabbit polyclonal anti-␤Tub56D (1:1000) was from Dr. Detlev Buttgereit (Philipps Universität Marburg, Germany) (Buttgereit et al, 1991) and rabbit polyclonal anti-dTau was from Dr. Nick Lowe (Cambridge University, UK) (1:2000). Appropriate HRP-conjugated secondary antibodies were applied at 1:5000.…”

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

“…In the Drosophila syncytial blastoderm, the anterior-posterior (AP) Bicoid gradient, perhaps the most well-studied morphogen gradient system, has long been thought to develop through a mechanism of diffusion from a spatially localized source (Driever and Nüsslein-Volhard, 1988;Houchmandzadeh et al, 2002;Gregor et al, 2005Gregor et al, , 2007Little et al, 2011). More recently, it has been proposed that the Bicoid gradient develops largely from a bicoid mRNA gradient, which itself developed through active transport (Spirov et al, 2009;Fahmy et al, 2014;Ali-Murthy and Kornberg, 2016).…”

Segmentation gene expression patterns in Bactrocera dorsalis and related insects: regulation and shape of blastoderm and larval cuticle

“…Templates for Riboprobes were generated using a 0-4 h B. dorsalis cDNA library (L. Ngernsiri, unpublished) as templates and T7 RNA polymerasebinding sites on the reverse primer from the above identified B. dorsalis segmentation genes. These DNA templates were purified, sequenced and used as templates using a DIG-labeling kit (Roche) as described (Fahmy et al, 2014). A non-related sense probe was used as a negative control.…”

“…For example, the maternal gene bicoid (bcd), shown to be a feature of higher Diptera only, is not present in the Bactrocera genome. In Drosophila, bicoid is expressed in a protein gradient along the A-P axis (Driever and Nüsslein-Volhard, 1988), preceded by the formation of a mRNA gradient (Frigerio et al, 1986;Spirov et al, 2009;Fahmy et al, 2014). The protein gradient serves as morphogen gradient to pattern the anterior-posterior axis.…”