Characterization of three spot, a mutation that disrupts germ cell migration of Drosophila melanogaster

During embryonic development, the somatic gonad precursors become populated by the primordial germ cells. While many genes have been identified, the highly regulated process of germ cell migration across the primordial midgut epithelium is not well characterized. Here, we describe three spot (tspt), a gene which interacts with tre1 during cross-epithelial migration. The tspt mutant was created in an EMS mutagenesis screen performed previously. Embryos from tspt mothers appear to exhibit restricted germ cell migration through the posterior midgut, resulting in a population of germ cells that remain associated with the endoderm through stage 16. We show that tspt is a maternally required migration gene, as well as a temperature sensitive mutant. We have identified two deletion regions that fail to complement tspt. One of the regions maps to the same region defined by recombination mapping. P-element insertions for 19 genes in and around this region were tested for germ cell phenotypes. Surprisingly, three of the nineteen exhibited germ cell defects. The Tre1 G-protein coupled receptor gene has been shown to be necessary for both programmed cell death and the migration of germ cells across the midgut. The scattershot allele of tre1 fails to complement tspt mutants, suggesting that they may be involved in the same or converging pathways during germ cell migration. Here we will describe the characterization of tspt mutants and the mapping of the tspt gene

Introductory biology undergraduate students\u27 mixed ideas about genetic information flow

The core concept of genetic information flow was identified in recent calls to improve undergraduate biology education. Previous work shows that students have difficulty differentiating between the three processes of the Central Dogma (CD; replication, transcription, and translation). We built upon this work by developing and applying an analytic coding rubric to 1050 student written responses to a three‐question item about the CD. Each response was previously coded only for correctness using a holistic rubric. Our rubric captures subtleties of student conceptual understanding of each process that previous work has not yet captured at a large scale. Regardless of holistic correctness scores, student responses included five or six distinct ideas. By analyzing common co‐occurring rubric categories in student responses, we found a common pair representing two normative ideas about the molecules produced by each CD process. By applying analytic coding to student responses preinstruction and postinstruction, we found student thinking about the processes involved was most prone to change. The combined strengths of analytic and holistic rubrics allow us to reveal mixed ideas about the CD processes and provide a detailed picture of which conceptual ideas students draw upon when explaining each CD process

Extradenticle and Homothorax Control Adult Muscle Fiber Identity in Drosophila

SummaryHere we identify a key role for the homeodomain proteins Extradenticle (Exd) and Homothorax (Hth) in the specification of muscle fiber fate in Drosophila. exd and hth are expressed in the fibrillar indirect flight muscles but not in tubular jump muscles, and manipulating exd or hth expression converts one muscle type into the other. In the flight muscles, exd and hth are genetically upstream of another muscle identity gene, salm, and are direct transcriptional regulators of the signature flight muscle structural gene, Actin88F. Exd and Hth also impact muscle identity in other somatic muscles of the body by cooperating with Hox factors. Because mammalian orthologs of exd and hth also contribute to muscle gene regulation, our studies suggest that an evolutionarily conserved genetic pathway determines muscle fiber differentiation

Somatic Mutagenesis with a Sleeping Beauty Transposon System Leads to Solid Tumor Formation in Zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers

A Hox Transcription Factor Collective Binds a Highly Conserved Distal-less cis-Regulatory Module to Generate Robust Transcriptional Outcomes.

cis-regulatory modules (CRMs) generate precise expression patterns by integrating numerous transcription factors (TFs). Surprisingly, CRMs that control essential gene patterns can differ greatly in conservation, suggesting distinct constraints on TF binding sites. Here, we show that a highly conserved Distal-less regulatory element (DCRE) that controls gene expression in leg precursor cells recruits multiple Hox, Extradenticle (Exd) and Homothorax (Hth) complexes to mediate dual outputs: thoracic activation and abdominal repression. Using reporter assays, we found that abdominal repression is particularly robust, as neither individual binding site mutations nor a DNA binding deficient Hth protein abolished cooperative DNA binding and in vivo repression. Moreover, a re-engineered DCRE containing a distinct configuration of Hox, Exd, and Hth sites also mediated abdominal Hox repression. However, the re-engineered DCRE failed to perform additional segment-specific functions such as thoracic activation. These findings are consistent with two emerging concepts in gene regulation: First, the abdominal Hox/Exd/Hth factors utilize protein-protein and protein-DNA interactions to form repression complexes on flexible combinations of sites, consistent with the TF collective model of CRM organization. Second, the conserved DCRE mediates multiple cell-type specific outputs, consistent with recent findings that pleiotropic CRMs are associated with conserved TF binding and added evolutionary constraints

DCRE activity does not require the Hth homeodomain.

<p>(A, B) <i>ArmG4;2xUD-lacZ</i> reporter activity visualized in Stage 11 embryos immunostained with β-gal (red) and Dll (cyan) in either <i>wild type</i> (A) or <i>hth</i>^{<i>100</i>.<i>1</i>} (homeodomain-lacking) (B) embryos demonstrates similar levels of β-gal expression. (C, D) <i>GD-lacZ</i> reporter activity visualized in Stage 15 embryos immunostained with β-gal (green) in either <i>wild type</i> (C) or <i>hth</i>^{<i>100</i>.<i>1</i>} embryos (D). (E) Quantification of β-gal intensity relative to T3 of <i>GD-lacZ</i> in <i>wild-type</i>, <i>hth</i>^{<i>100</i>.<i>1</i>}, and <i>hth</i>^<i>p2</i> (strong hypomorph) embryos demonstrates that <i>GD-lacZ</i> repression requires Hth, but not the Hth homeodomain.</p

Hox binding to linked Hox/Exd and Hox/Hth sites within the DCRE.

<p>(A-I) EMSAs performed on the DCRE full-length probe (for sequence, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005981#pgen.1005981.g001" target="_blank">Fig 1A</a>). (A-C) Titration of Antp protein (concentrations from 37.5 nM to 300 nM) alone (A), with 25 nM purified Exd/Hth dimer (B), or with 200 nM purified Exd/HthΔHD dimer (C). (D-I) Titration of AbdA protein (concentrations from 37.5 nM to 300 nM) on the DCRE wild-type probe alone or with (D) 75 nM purified Exd/Hth dimer (E) 200 nM purified Exd/HthΔHD dimer (F), 200nM purified ExdN51A/Hth (G), 200 nM purified Exd (H), or 200 nM purified Hth (I). * indicates Hox-only binding to DCRE. Arrow indicates higher order complex seen with AbdA/Exd/Hth but not Antp/Exd/Hth. Filled arrowheads indicate Hth/Exd heterodimer binding. Empty arrowheads indicate lack of binding by Hth and/or Exd.</p

The abdominal Hox factors repress <i>Distal-less</i> via DCRE-dependent and–independent mechanisms.

<p>(A) Schematic of the DMX enhancer containing the DMEact and the DCRE. Detail shows the DCRE sequence with known TF binding sites (FoxG, Hox1, Exd, En, Hth, and Hox2) highlighted. Note, the Exd0 and Hox0 sites are new sites characterized in this manuscript. (B) <i>DMX-lacZ</i> is expressed in Dll-positive (Dll+) cells of the thorax. (C) <i>DMEact-lacZ</i> is expressed in Dll+ cells of the thorax and the corresponding cells of the abdomen. (D-E) Quantification of β-gal expression area (D) and β-gal intensity relative to Dll expression in the same embryos (E) in <i>DMX-lacZ</i> and <i>DMEact-lacZ</i> embryos demonstrates that the DMEact is not fully de-repressed in the abdomen and has reduced thoracic levels when compared to thoracic levels of DMX. (F-I) Effect of Hox gene mis-expression on reporter activity in <i>PrdG4;UAS-AbdA;DMX-lacZ</i> (F), <i>PrdG4;UAS-AbdA;DMEact-lacZ</i> (G), <i>PrdG4;UAS-Ubx;DMX-lacZ</i> (H), and <i>PrdG4;UAS-Ubx;DMEact-lacZ</i> (I) embryos. (J) Quantification of β-gal levels in T2 mis-expressing segments relative to non-Gal4 expressing control T3 segments shows that both Abd-A and Ubx repress via DCRE-dependent and -independent mechanisms. All images are lateral views of Stage 11 embryos immunostained for β-gal (red or white) and Dll, Abd-A, or Ubx (cyan) as indicated. (Statistics ** p < 0.01, Welch’s t-test, error bars S.E.M). Note, as a control for the PrdG4 experiments, we quantified the levels of βgal in the absence of Gal4 and noted no significant differences between the T2 vs T3 segments or the A1 vs A2 segments of <i>DMX-lacZ</i> and <i>DMEact-lacZ</i> embryos. (DMX β-gal pixel intensity: Thorax, T2 = 113 ± 13%, T3 = 112 ± 10%, Abdomen, A1 = 44 ±10%, A2 = 56 ± 13%. DMEact β-gal pixel intensity: Thorax, T2 = 98 ± 12%, T3 = 96 ± 11, Abdomen, A1 = 69 ±12%, A2 = 70 ± 16%).</p

The DCRE is sufficient for repression in a cell-specific manner in the abdomen.

<p>(A) Schematics of the <i>GBE-lacZ</i> and <i>GD-lacZ</i> reporter assays. (B, C) <i>GBE-lacZ</i> embryos reveal relatively uniform β-gal expression in posterior En+ cells (B) and anterior Slp2+cells (C). (D, E) <i>GD-lacZ</i> embryos reveal that the DCRE represses gene expression in Slp2+ (E) but not En+ (D) abdominal cells. (F) Quantification of β-gal intensity of thoracic and abdominal segments relative to segment T3 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005981#sec013" target="_blank">Materials and Methods</a>) for <i>GD-lacZ</i> activity in Slp2+ versus Slp2- cells demonstrates that the DCRE represses gene expression in Slp+ cells of the abdomen. (G) Quantification of β-gal intensity of thoracic and abdominal segments of <i>GD-lacZ</i> versus <i>GBE-lacZ</i>. All images are lateral views of Stage 15 embryos immunostained for β-gal (green or white) and Slp2 or En (magenta) as indicated. (Statistics <b>*</b> p < 0.05, n.s. not significant, Welch’s t-test, Error Bars S.E.M).</p

Homothorax and Sloppy-paired are genetically required for DCRE-mediated repression.

<p>(A-C) <i>GD-lacZ</i> activity in <i>wt</i> (A), <i>Slp</i>^<i>Δ34</i> (B) and <i>hth</i>^<i>P2</i> (C) embryos reveals that Slp and Hth are required for DCRE-repression. All images are lateral views of Stage 15 embryos immunostained for β-gal (green or white) and AbdA or Slp (magenta) as indicated.</p