Novel Behavioral and Developmental Defects Associated with Drosophila single-minded

In Drosophila, the development of the midline cells of the embryonic ventral nerve cord depends on the function of the bHLH-PAS transcription factor Single-minded (Sim). The expression domain of sim, however, is also found anterior and posterior to the developing ventral cord throughout the germ band. Indeed, mutations in sim were identified based on their characteristic cuticle phenotype. Eight abdominal segments (A1–A8) can be easily seen in the larval cuticle, while three more can be identified during embryogenesis. Cells located in A8–A10 give rise to the formation of the genital imaginal discs, and a highly modified A11 segment gives rise to the anal pads that flank the anus. sim is expressed in all these segments and is required for the formation of both the anal pads and the genital imaginal discs. A new temperature-sensitive sim allele allowed an assessment of possible postembryonic function(s) of sim. Reduction of sim function below a 50% threshold leads to sterile flies with marked behavioral deficits. Most mutant sim flies were only able to walk in circles. Further analyses indicated that this phenotype is likely due to defects in the brain central complex. This brain region, which has previously been implicated in the control of walking behavior, expresses high levels of nuclear Sim protein in three clusters of neurons in each central brain hemisphere. Additional Sim localization in the medullary and laminar neurons of the optic lobes may correlate with the presence of ectopic axon bundles observed in the optic lobes of sim mutant flies

Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA[superscript Gly] aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.National Institutes of Health (U.S.) (Grant GM17151

Genetische Kontrolle axonaler Konvergenz und synaptischer Spezifität olfaktorischer Rezeptorneurone in Drosophila melanogaster

Das olfaktorische System von Drosophila wurde als Modellsystem zur systematischen Untersuchung von Genen, die an der Ausbildung der synaptischen Spezifität beteiligt sind, ausgewählt. In dieser Arbeit wurden mittels eines systematischen genetischen Ansatzes ca. 1400 Letalmutationen in einer Mosaikanalyse auf Defekte in der Entwicklung olfaktorischer Rezeptorneurone (ORNs) gesichtet. Es konnten 52 Mutationen identifiziert werden, die zu Verschaltungsdefekten im olfaktorischen System führen. In 22 Mutationen konnte die Letalität einer zytologischen Region zugewiesen werden und 15 dieser 22 Mutationen konnten einem Gen bzw. einer Komplementationsgruppe zugeordnet werden. Die Rolle des Enzyms Serin-Palmitoyltransferase in der axonalen Konvergenz und die Funktion des Transkriptionsregulators Posterior sex combs in der synaptischen Spezifität wurde genauer untersucht

Solar Electric Propulsion Module Concept for the BiFrost Architecture

IAC-02-S.4.09, 53rd International Astronautical Congress, The World Space Congress Houston, TX, October 10-19, 2002.This paper describes the design of a solar electric propulsion module for the Bifrost architecture. Bifrost consists of a magnetic levitation launch tube with the exit end elevated to 20 km. A 35,000 kg hybrid logistics module (HLM) is designed to attach to an array of propulsion modules that accommodate different missions. The solar electric propulsion (SEP) module is designed to circularize a payload in Geosynchronous Earth orbit (GEO) from a highly elliptic transfer orbit. A configuration consisting of a central spacecraft body propelling itself with electric thrusters and gathering solar power from two inflatable concentrating reflectors was chosen. Concentrating reflectors were chosen over thin film arrays due to the large mass savings. Details of the conceptual design process are presented. Disciplines include trajectory, power system, propulsion, and weights & sizing. A computational framework was used to wrap the disciplinary analysis to speed the design process, and optimization was performed to minimize the initial mass of the vehicle from within the design framework. The resulting vehicle has an initial mass in orbit of 40,780 kg. A demonstration model was then designed and constructed from the conceptual design. The manufacturing process for the inflatable reflector and the spacecraft body are described in detail. The demonstration model shows that an inflatable reflector is a feasible method of generating large amounts of power in space

Mutanlallemand (<em>mtl</em>) and Belly Spot and Deafness (<em>bsd</em>) Are Two New Mutations of <em>Lmx1a</em> Causing Severe Cochlear and Vestibular Defects

<div><p><em>Mutanlallemand</em> (<em>mtl</em>) and <em>Belly Spot and Deafness</em> (<em>bsd</em>) are two new spontaneous alleles of the <em>Lmx1a</em> gene in mice. Homozygous mutants show head tossing and circling behaviour, indicative of vestibular defects, and they have short tails and white belly patches of variable size. The analysis of auditory brainstem responses (ABR) showed that <em>mtl</em> and <em>bsd</em> homozygotes are deaf, whereas heterozygous and wildtype littermates have normal hearing. Paint-filled inner ears at E16.5 revealed that <em>mtl</em> and <em>bsd</em> homozygotes lack endolymphatic ducts and semicircular canals and have short cochlear ducts. These new alleles show similarities with <em>dreher</em> (<em>Lmx1a</em>) mutants. Complementation tests between <em>mtl</em> and <em>dreher</em> and between <em>mtl</em> and <em>bsd</em> suggest that <em>mtl</em> and <em>bsd</em> are new mutant alleles of the <em>Lmx1a</em> gene. To determine the <em>Lmx1a</em> mutation in <em>mtl</em> and <em>bsd</em> mutant mice we performed PCR followed by sequencing of genomic DNA and cDNA. The <em>mtl</em> mutation is a single point mutation in the 3′ splice site of exon 4 leading to an exon extension and the activation of a cryptic splice site 44 base pairs downstream, whereas the <em>bsd</em> mutation is a genomic deletion that includes exon 3. Both mutations lead to a truncated LMX1A protein affecting the homeodomain (<em>mtl</em>) or LIM2-domain (<em>bsd</em>), which is critical for LMX1A protein function. Moreover, the levels of <em>Lmx1a</em> transcript in <em>mtl</em> and <em>bsd</em> mutants are significantly down-regulated. <em>Hmx2/3</em> and <em>Pax2</em> expression are also down-regulated in <em>mtl</em> and <em>bsd</em> mutants, suggesting a role of <em>Lmx1a</em> upstream of these transcription factors in early inner ear morphogenesis. We have found that these mutants develop sensory patches although they are misshapen. The characterization of these two new <em>Lmx1a</em> alleles highlights the critical role of this gene in the development of the cochlea and vestibular system.</p> </div

Deletion of exon 3 of <i>Lmx1a</i> in <i>bsd</i> mutants. A

<p>, PCR amplification of genomic DNA from +/+, +/<i>bsd</i> and <i>bsd</i>/<i>bsd</i> mice with primers specific to each exon of <i>Lmx1a</i> gene. One single band is amplified for each exon and sequenced for all <i>bsd</i> genotypes with no differences between mutants (M) and controls (wt, het) except for exon 3, where no band was amplified in <i>bsd</i> mutants (asterisk). <b>B</b>, Partial traces and sequence of <i>Lmx1a</i> intron 4–5 are shown. At 30 base pairs downstream of exon 4 we identified a single nucleotide polymorphism (SNP)-from A to G- that we used to genotype <i>bsd</i> mice. <b>C</b>, PCR amplification of <i>bsd</i> cDNA from +/+, +/<i>bsd</i> and <i>bsd</i>/<i>bsd</i> mice at E10.5 with primers specific to transcript Lmx1a-001 covering exons 2–8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051065#pone.0051065.s004" target="_blank">Table S2</a>). Bands were amplified for controls (wt, het) but no band was detected in mutants (M). We designed primers for small fragments of the transcript (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051065#pone.0051065.s004" target="_blank">Table S2</a>). Controls (+/+) showed bands for all combinations of primers (numbers on top indicate position of primers forward and reverse, respectively) whereas in <i>bsd/bsd</i> no bands were amplified by primers 13 and 35 (black asterisks). Interestingly, we found a small band of less than 100 bps with primers 24. This band is likely to correspond to the size of the amplicon (322 bp) minus the deleted exon 3 (233 bp) of <i>bsd</i> mutants. <b>D</b>, Diagram showing structure of <i>Lmx1a</i> gene in +/+, <i>mtl</i>/<i>mtl</i> and <i>bsd</i>/<i>bsd</i>. Initiation codon in exon 1 and termination codon in exon 8 are indicated. Point mutation in <i>mtl</i> homozygotes is indicated by a red asterisk and deletion in <i>bsd</i> mutants is shown by a red dotted line. <i>Mtl</i> mutation affects the homeodomain whereas in <i>bsd</i> the deletion of exon 3 involves the LIM2 domain.</p

Schuermann_Figure_3

Schuermann_Figure_3 - contains Excel-data on HhC85S multimerization and demonstrates that HhNC85S, a non-lipidated artificial Hh variant, does not act in dominant-negative manner. Contains Prize-Files to quantify unaffected wing patterning in three independently derived HhNC85S lines. The original wing patterns are shown in folders en>HhNC85S and ptc>HhNC85S

Data from: Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions

Expression analysis of <i>Lmx1a</i>, FOXI1 and FGF9 in the developing inner ear. A–F

<p>, <i>In situ</i> hybridization of <i>Lmx1a</i> on wildtype inner ear sections at E10.5 (<b>A, C, E</b>) and E12.5 (<b>B, </b><b>D, F</b>). <i>Lmx1a</i> mRNA expression is shown in blue and nuclear counterstain in pink. In wildtype inner ears <i>Lmx1a</i> is expressed in the otic vesicle and in the endolymphatic sac (black arrow in <b>A, </b><b>B</b> and <b>D</b>), whereas delaminating neuroblasts are <i>Lmx1a-negative</i> (<b>A, </b><b>C, </b><b>E,</b> white arrows). At E12.5 <i>Lmx1a</i> expression is also found in the region of the fusion plates which will later form the semicircular canals (asterisk in <b>B</b>). <i>Lmx1a</i> mRNA expression appears to be restricted to prospective non-sensory inner ear tissue. <b>G–H</b>, In wildtypes FOXI1 is specifically expressed in a subpopulation of cells within the endolymphatic sac (<b>G</b>) whereas in <i>mtl</i> mutants the typical evagination of the endolymphatic sac/duct fails and no FOXI1-positive cells are detected in or adjacent to the otic vesicle/otocyst (<b>H</b>). <b>I–S</b>, At E11.5 FGF9 expression highlights areas of epithelial thickening on the luminal as well as the mesenchymal side of the otic vesicle epithelium, such as the outpocketings of the developing semicircular canals (arrows in <b>I</b>). The endolymphatic duct is negative for FGF9 (asterisk in <b>I</b>). <b>J–S</b>, Series of sections covering the complete otocyst arranged from lateral to medial, of one <i>mtl</i> homozygote showing only a rudimentary otocyst epithelium rearrangement. Sections also show complete absence of an endolymphatic duct/sac structure. FGF9 is expressed in the mutant epithelium and marks epithelial thickening, but unlike in wildtypes there is no semicircular canal formation. The luminal and mesenchyme sides of the thickened mutant epithelium looked underdeveloped (arrows in <b>I</b> and <b>P</b>). SG, spiral ganglion. Scale bars: 100 µm.</p