Distinct molecular strategies for Hox-mediated limb suppression in Drosophila: from cooperativity to dispensability/antagonism in TALE partnership

International audienceThe emergence following gene duplication of a large repertoire of Hox paralogue proteins underlies the importance taken by Hox proteins in controlling animal body plans in development and evolution. Sequence divergence of paralogous proteins accounts for functional specialization, promoting axial morphological diversification in bilaterian animals. Yet functionally specialized paralogous Hox proteins also continue performing ancient common functions. In this study, we investigate how highly divergent Hox proteins perform an identical function. This was achieved by comparing in Drosophila the mode of limb suppression by the central (Ultrabithorax and AbdominalA) and posterior class (AbdominalB) Hox proteins. Results highlight that Hox-mediated limb suppression relies on distinct modes of DNA binding and a distinct use of TALE cofactors. Control of common functions by divergent Hox proteins, at least in the case studied, relies on evolving novel molecular properties. Thus, changes in protein sequences not only provide the driving force for functional specialization of Hox paralogue proteins, but also provide means to perform common ancient functions in distinct ways

Antagonism versus cooperativity with TALE cofactors at the base of the functional diversification of Hox protein function

International audienceExtradenticle (Exd) and Homothorax (Hth) function as positive transcriptional cofactors of Hox proteins, helping them to bind specifically their direct targets. The posterior Hox protein Abdominal-B (Abd-B) does not require Exd/Hth to bind DNA; and, during embryogenesis, Abd-B represses hth and exd transcription. Here we show that this repression is necessary for Abd-B function, as maintained Exd/Hth expression results in transformations similar to those observed in loss-of-function Abd-B mutants. We characterize the cis regulatory module directly regulated by Abd-B in the empty spiracles gene and show that the Exd/Hth complex interferes with Abd-B binding to this enhancer. Our results suggest that this novel Exd/Hth function does not require the complex to bind DNA and may be mediated by direct Exd/Hth binding to the Abd-B homeodomain. Thus, in some instances, the main positive cofactor complex for anterior Hox proteins can act as a negative factor for the posterior Hox protein Abd-B. This antagonistic interaction uncovers an alternative way in which MEIS and PBC cofactors can modulate Abd-B like posterior Hox genes during development

Membrane and synaptic defects leading to neurodegeneration in Adar mutant Drosophila are rescued by increased autophagy

BackgroundIn fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar5G1 null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration.ResultsA genetic screen revealed suppression of all Adar5G1 mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar5G1 phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar5G1 mutant phenotypes tested.ConclusionsThese findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage

Identification of a novel target of D/V signaling in Drosophila wing disc: Wg-independent function of the organizer

Growth and patterning during Drosophila wing development are mediated by signaling from its dorso-ventral (D/V) organizer. Wingless is expressed in the D/V boundary and functions as a morphogen to activate target genes at a distance. Wingless pathway and thereby D/V signaling is negatively regulated by the homeotic gene Ultrabithorax (Ubx) to mediate haltere development. In an enhancer-trap screen to identify genes that show differential expression between wing and haltere discs, we identified CG32062, which codes for a RNA-binding protein. In wing discs, CG32062 is expressed only in non-D/V cells. CG32062 expression in non-D/V cells is dependent on Notch-mediated signaling from the D/V boundary. However, CG32062 expression is independent of Wingless function, thus providing evidence for a second long-range signaling mechanism of the D/V organizer. In haltere discs, CG32062 is negatively regulated by Ubx. The non-cell autonomous nature of Ubx-mediated repression of CG32062 expression suggests that the novel component of D/V signaling is also negatively regulated during haltere specification

Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch

International audienceTranslational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31B•Tral complex may be at the core of repression

Protein sequence requirements for AbdB-mediated DME repression.

<p>Sequence conservation in the AbdB HD (shown only for the N-terminal arm and helix 3) and HD flanking regions HX/LR and C-ter region. Sequences upstream and downstream of these regions display progressively weaker conservation. Web logo was obtained using sequences the following AbdB sequences (Drosophila (AAA84402), Tribolium (AAF36721.1), Anophela (XM311628), Sacculine (AAQ49317.1), Folsomia (AAK52499.1) and human (BCO10023)). The <i>Drosophila melanogaster</i> sequence is shown below the web logo. The position and the nature of the mutations generated are represented below the web logo. Effects of the mutations on the repressive activity of AbdB on DME are displayed in a box plot representation. While mutations in the HX/LR region have little effect on AbdB repressive activity, mutations within the HD, including the N-terminal arm, helix 3 and the Cter alter to different extent AbdB repressive potential. Illustration of data is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307.s010" target="_blank">Figure S10</a>.</p

AbdB m and r isoforms repress <i>Dll</i> in the posterior abdominal segments A8 and A9.

<p>(A) Embryo stained for β-gal driven by the DMX enhancer (red) showing restricted thoracic activity. (B–C) DMX(X2X5) embryos co-stained for β-gal (red) and AbdB (green). This modified DMX enhancer is derepressed in all abdominal segments, including A8 and A9. Co-localization of β-gal and AbdB in A8 and A9 cells normally subject to enhancer activity repression is highlighted in the magnified view. (D–E) Embryo lacking Ubx and AbdA function (<i>Df(109)</i>) shows DMX abdominal de-repression (red) up to A7 (arrowhead) and remains repressed in A8 and A9 segments where AbdB is expressed at high levels (green). (F–G) Embryo lacking Ubx, AbdA and the AbdBm isoform, but retaining the AbdB r isoform, shows derepression of DMX (red) till A8 (arrowhead). (H–I) Embryos lacking Ubx, AbdA and the AbdB m and r isoforms (<i>Df P9</i>) show DMX de-repression (red) in all abdominal segments, including in A9 (arrowhead). (J–K) <i>prd-Gal4</i> driven ectopic expression of the AbdBm isoform represses DMX activity (arrow in T2). (L–M) <i>prd-Gal4</i> driven ectopic expression of the AbdBr isoform represses DMX activity (arrow in T2).</p

Requirement of protein domains in AbdB/Ubx chimeric proteins for DME repression.

<p>Left part of the figure depicts wild type and mutated variants of Ubx (blue), including mutations (indicated by crosses) in the HX, and UbdA (UA) domains as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307-Merabet2" target="_blank">[15]</a> and in helix 3 (Q50 to K50) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307-Chan1" target="_blank">[55]</a>. AbdB protein sequences, including or not the QR domain, either with a wild type or mutated helix 3, is represented in red. Effects of the mutations on the repressive activity of AbdB on DME are displayed in a box plot representation. Switching the Ubx HD by that of AbdB endows the chimera with a posterior AbdB like dependent mode of DME repression. Illustration of data is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307.s011" target="_blank">Figure S11</a>.</p

Models for distinct Hox cofactor partnership for <i>Dll</i> repression.

<p>(A) Model for repression of <i>Dll</i> by Ubx/AbdA in anterior abdominal segments A1-7. Repression relies on the assembly of a Hox/Exd/Hth protein complex <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307-Gebelein2" target="_blank">[20]</a>. DNA binding by Hox proteins is not essential (depicted by a dashed delineated pink zone of contact between the Hox protein and the DNA), as supported by the limited loss of repressive activity of a DNA binding deficient Ubx protein (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen-1003307-g007" target="_blank">Figure 7</a>), and by the limited dereprepression associated to mutation in Hox binding sites. The non-essential character of Hox DNA binding may result from acting in a context of a multiprotein complex containing two additional DNA binding proteins (Exd and Hth). (B) Model for repression of <i>Dll</i> by AbdB in posterior abdominal segments in A8-9. AbdB represses Exd and Hth, and consequently act without the aid of Exd and Hth to repress <i>Dll</i>. This difference likely imposes a strict requirement for AbdB DNA binding for efficient repression.</p