Functional characterization of epididymal protein DE (CRISP-1) through in vitro studies and generation of "knock out" animals

La proteína epididimaria de rata DE es el primer miembro descripto de la familia de proteínas ricas en cisteínas (CRISP) y, por ende, también conocida como CRISP-1. DE se asocia a la región dorsal de la cabeza del espermatozoide durante la maduración epididimaria, migra al segmento ecuatorial bajo condiciones capacitantes, y participa en el proceso de fusión de gametas a través de sitios complementarios presentes en la superficie del ovocito. El primer objetivo de este trabajo fue estudiar la influencia del bicarbonato, ión indispensable para la capacitación, sobre la migración de la proteína DE. En paralelo, se evaluó el efecto sobre otros dos parámetros espermáticos asociados a la capacitación, tales como la fosforilación de proteínas en tirosina y la expresión de la capacidad fusogénica. Los resultados indicaron que si bien los tres parámetros evaluados fueron bicarbonatodependientes, la migración de DE al segmento ecuatorial ocurriría a través de vías alternativas a la clásica cAMP/PKA, involucrada en la fosforilación en tirosina y la capacidad fusogénica del espermatozoide. El segundo objetivo de este trabajo consistió en estudiar los mecanismos moleculares por los cuales DE interactúa con sus sitios en el ovocito. El empleo de fragmentos recombinantes y péptidos sintéticos indicó que el sitio activo de DE residiría en una región de sólo 12 aminoácidos altamente conservada en todos los miembros de la familia CRISP. Finalmente, la relevancia de DE/CRISP-1 para la fertilidad fue estudiada a través de la generación de ratones “knock out” para dicha proteína. El análisis del fenotipo de los mismos indicó que si bien la fertilidad de los animales no se vio afectada, los espermatozoides presentaban una capacidad fusogéncia significativamente disminuida, sugiriendo que otras proteínas con características y/o función similares estarían compensando el efecto observado. En conjunto, estos resultados proveen importante información sobre los mecanismos moleculares involucrados en la interacción de gametas y apoyan la existencia de mecanismos compensatorios con el fin de asegurar el éxito de la fertilización.Rat epididymal protein DE is the first described member of the cysteine rich secretory protein family (CRISP) and, thus also known as CRISP-1. DE associates with the dorsal region of the sperm head during epididymal maturation, migrates to the equatorial segment under capacitanting conditions and participates in gamete fusion through complementary sites on the egg surface. The first aim of this work was to study the influence of bicarbonate, essential anion for sperm capacitation, on DE migration. In parallel, we investigated the influence of bicarbonate on two other sperm functional parameters associated with capacitation, such as tyrosine protein phosphorylation and expression of the fusogenic ability. Results indicated that although the three parameter were bicarbonate-dependent, DE migration would occur through a signaling pathway alternative to the classic cAMP/PKA involved in tyrosine phosphorylation and expression of the fusogenic ability. The second objective of the present work was to study the molecular mechanisms involved in the interaction between DE and its complementary sites on the egg surface. The use of recombinant and synthetic peptides indicated that the active site of DE resides in a region of only 12 aminoacids highly conserved among members of the CRISP family. Finally, the relevance of DE/CRISP-1 for animal fertility was evaluated through the generation of knock out animals for this protein. The analysis of the phenotype of these animals showed that, although their fertility was not affected, the sperm fusogenic ability was significantly reduced, suggesting that other proteins with similar characteristics and/or function, would be compensating the described effect. Altogether, these results provide important information on the molecular mechanisms involve in gamete interaction, and support the existence of compensatory mechanisms to ensure the success of fertilization.Fil: Da Ros, Vanina G.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Dampening the signals transduced through hedgehog via microRNA miR-7 facilitates notch-induced tumourigenesis.

Fine-tuned Notch and Hedgehog signalling pathways via attenuators and dampers have long been recognized as important mechanisms to ensure the proper size and differentiation of many organs and tissues. This notion is further supported by identification of mutations in these pathways in human cancer cells. However, although it is common that the Notch and Hedgehog pathways influence growth and patterning within the same organ through the establishment of organizing regions, the cross-talk between these two pathways and how the distinct organizing activities are integrated during growth is poorly understood. Here, in an unbiased genetic screen in the Drosophila melanogaster eye, we found that tumour-like growth was provoked by cooperation between the microRNA miR-7 and the Notch pathway. Surprisingly, the molecular basis of this cooperation between miR-7 and Notch converged on the silencing of Hedgehog signalling. In mechanistic terms, miR-7 silenced the interference hedgehog (ihog) Hedgehog receptor, while Notch repressed expression of the brother of ihog (boi) Hedgehog receptor. Tumourigenesis was induced co-operatively following Notch activation and reduced Hedgehog signalling, either via overexpression of the microRNA or through specific down-regulation of ihog, hedgehog, smoothened, or cubitus interruptus or via overexpression of the cubitus interruptus repressor form. Conversely, increasing Hedgehog signalling prevented eye overgrowth induced by the microRNA and Notch pathway. Further, we show that blocking Hh signal transduction in clones of cells mutant for smoothened also enhance the organizing activity and growth by Delta-Notch signalling in the wing primordium. Together, these findings uncover a hitherto unsuspected tumour suppressor role for the Hedgehog signalling and reveal an unanticipated cooperative antagonism between two pathways extensively used in growth control and cancer

Downregulation of elements in the Hh pathway or overexpression of the repressor form of <i>ci</i> co-operates with <i>Dl</i> overexpression to trigger tumour growth in the <i>Drosophila</i> eye.

<p>(A) Schematic representation of Hh signalling and the <i>UAS</i> transgenes used to downregulate by RNAi (IR) or activate Hh pathway components. (B–D, H, and K–L) Representative adult heads of female flies of combinations of the indicated <i>UAS</i> transgenes and <i>ey-Gal4</i> are shown. (E–F) Fluorescent images of <i>Drosophila</i> pupae of sibling control (<i>ey>Dl</i>, E) or <i>ey>Dl>ci-IR</i> (F). (G) Adult fly of <i>ey>Dl>ci-IR</i> with a metastatic (met) growth in the abdomen. Eye tissue in the endogenous site (green arrowheads) and distant site (white arrowheads) is labelled by the retinal-specific GMR-myrRFP marker (E, F) or the retinal-specific red pigments (G). (I–J′) Third instar wild type of sized eye disc (I) and <i>ey>Dl</i> eye disc carrying clones of <i>hh^AC</i> labelled by the absence of <i>arm-lacZ</i> (ßgal, red in J and grey in J′). Arrowhead points to a clone and its associated twin spot (high red staining). (M) Model of antagonistic interaction between Hh and Notch signalling in normal eye imaginal disc (left) and model of regulatory interactions among the microRNA, Notch pathway, and the Hh receptors <i>ihog</i> and <i>boi</i> (right). Genotype in (J) is: <i>yw ey-Flp; ey-Gal4 UAS-Dl/+; FRT82B hh^AC/FRT82B arm-lacZ</i>.</p

The Conserved MicroRNA miR-7 co-operates with Notch in <i>D. melanogaster</i> oncogenesis.

<p>(A) A schematic outline of the <i>Gene Search</i> (<i>GS</i>) gain-of-expression screen for Notch co-operating oncogenes in the developing <i>Drosophila</i> eye. (B–E and K–L) Adult heads of control female <i>ey-Gal4</i> wild-type eye size (B) and combinations between GS line, UAS transgenes, and <i>ey-Gal4</i> are shown. (C) <i>Dl</i> expression under the control of <i>ey-Gal4</i> results in a mild overgrowth in the eye (130% larger than wild type size). (D) Introducing the <i>GS(2)518ND2</i> line enhanced overgrowth by <i>Dl</i> (>320%, see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001554#pbio.1001554.s002" target="_blank">Figure S2</a>). (E) The overexpression of gene(s) affected by the <i>GS(2)518ND2</i> line alone causes no overt eye overgrowth. (F) Scheme of the <i>GS(2)518ND2</i> insertion. (G) Overexpression of the <i>GS(2)518ND2</i> line driven by <i>ptc-Gal4</i> showed the typical wing vein L3–L4 fusion. (H–I′) Confocal images of third instar eye-antennal discs stained for the mitotic marker PH3 (red), Wg (blue) to define the DV axis, and the neuronal marker Elav (green) of the indicated genotypes. White arrowheads indicate the position of the MF. The co-expression of <i>UAS-mir-7</i> with <i>UAS-Dl</i> causes eye disc overgrowth and a front of retinal differentiation highly disorganized (H, compare with control sibling eye disc in I). (J) Quantification of mitotic cells labelled by PH3 anterior to the MF of the genotypes: <i>ey>Dl>mir-7</i> (red bar), <i>ey>Dl</i> (green bar), and wild-type sibling discs +/<i>UAS-mir7</i> (<i>>mir-7</i>, blue bar). Data shown represent the mean ± s.e.m. of total PH3 measurement in 20 eye discs per genotype. <i>P</i> values were calculated by the unpaired Student's <i>t</i> test. (K–L) Adult heads overexpressing <i>mir-7</i> driven by <i>ey-Gal4</i> in the presence (K) or the absence (L) of the <i>UAS-Dl</i> transgene. See also Figures S2 to S4 for supplementary data.</p

miR-7 silencing of Hh signalling explains the L3–L4 fusion defects in the wing.

<p>(A) Ci protein (green) is distributed across the entire anterior (A) compartment of the discs. Hh signals from posterior (P) cells induce high levels of Ci in cells along the AP border, and they block Ci proteolysis into the repressor form (Ci[rep]), thereby allowing the Ci activator (C[act]) to accumulate. (B–B′) Overexpression of <i>mir-7</i> denoted by red labelling (<i>UAS-DsRed::mir-7</i>) driven by <i>patched (ptc)-Gal4</i> downregulates Hh signalling as visualized by low Ci levels (green; white arrowhead). Insets show magnifications. Engrailed (En) staining in blue serves to mark the P compartment in (A–B″). Plots of fluorescence intensity profiles of the anterior-posterior compartments from the WT (A) and <i>ptc>DsRed::mir-7</i> (B′) discs are shown in (A′) and (B″), respectively. Green trace, Ci; blue trace, En; red trace, DsRed. (C) Adult wild-type wing. The shaded area denotes the domain of expression of the <i>ptc-Gal4</i> reporter. (D) <i>ci-IR</i> expression by <i>ptc-Gal4</i> mimicking the L3–L4 fusion defect seen in adult wings that is caused by <i>mir-7</i> overexpression (compare with <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001554#pbio-1001554-g001" target="_blank">Figure 1G</a>). (E) Adult wing expressing <i>ihog-IR</i> driven by <i>ptc-Gal4</i>. (F–F″) The expression of <i>boi-lacZ</i> (green) defines all longitudinal veins (L2–L5). Note the high <i>boi-</i>lacZ (green in F) expression along L3, marked by high Ci (red in F) and Dl (magenta in F″). (G–G″) Overexpression of <i>mir-7</i> (in red) by <i>Bx-Gal4</i> did not alter Ci protein levels (green, white arrowhead).</p

Notch signalling represses <i>brother of ihog</i> (<i>boi</i>) expression in the dorsal-ventral growth organizer in <i>Drosophila</i> eye.

<p>(A–B) Adult heads of female flies overexpressing <i>UAS-Dl</i> and/or <i>UAS-boi-IR</i> and <i>ey-Gal4</i>. (C) Map of <i>PlacW10111 P</i>-element insertion (triangle) into the <i>boi</i> locus. (D–I) <i>boi</i> expression in wild-type (D, E, F, and H) and Notch pathway mutant (G and I) eye-antennal discs. The patterning gene wingless (a-Wg, in red) serves to orient the eye disc in the dorsal (D)/ventral (V) axis. Expression of Boi (green) Hh co-receptor at the early third larval stage is repressed along the DV organizer (D and E), as defined by the expression of the DV organizer gene <i>eyg</i> (blue, E and F). Retinal differentiation (neuronal marker a-Elav, magenta) is first detected at the posterior end of the eye disc (to the right) and progresses in an anterior direction (H). The arrow points to the MF. (G and I) Expression of <i>boi-lacZ</i> (<i>boi-Z</i>, green) and wingless (a-Wg, red) in <i>ey>eyg</i> (G) and <i>ey>fng</i> (I) eye discs. The discs in (H) and (I) are from the same stage and magnification. The enlarged antennal disc in (I) is an effect of the undergrowth of the eye disc, caused in part by defective Notch activation in the D/V organizer due to <i>fng</i> overexpression. (J) qRT-PCR analyses of <i>boi</i> (left) and <i>ihog</i> (right) in <i>ey-Gal4</i> (white bar), <i>ey>Dl</i> (blue bar), and <i>ey>Dl>mir-7</i> (red bar) late third instar eye discs. Two independent experiments of three replicates are shown in each case. Data were normalized to <i>rp49</i>. mRNA isolated from 50 pairs of eye-antennal discs per genotype. Data analysed by a two-tailed unpaired <i>t</i> test. Error bars represent s.e.m. of three replicates. (K) Adult fly head showing no eye overgrown induced by <i>Dl</i> and <i>mir-7</i> when <i>boi</i> is overexpressed by a transgene (<i>UAS-boi</i>, 100% penetrance of rescue).</p

Failure to transduce the Hh signal due to mutations in smoothened enhances Dl-Notch signalling activity in the wing.

<p>(A–B″) Confocal images of wing discs bearing MARCM GFP (green)-labelled clones homozygous for <i>smo³</i> without (A) or with (B) <i>Dl</i> overexpression. Single channel images are also shown. Mosaic discs were stained for Wg (red in A and B, and grey in A′ and B′), and Ci (blue) and Ptc-lacZ (Ptc-Z, blue). (C) A schematic summary of clones in (B). Asterisks in (A″) and (B″) point to “posteriorly” situated clones that were of anterior origin as denoted by the failure to induce Ptc and the low levels of Ci protein (white line delineates the AP boundary in the discs in B). Clones were generated at 24–42 h after egg laying (AEL) by a 1 h heat shock at 37°C (<i>n = </i>60 clones analysed). Genotypes: (A) <i>yw hsp70-Flp tub-G4 UAS-GFP; tub-Gal80 FRT40A/smo³ FRT40A ptc-lacZ</i> and (B) <i>yw hsp70-Flp Tub-G4 UAS-GFP; Tub-Gal80 FRT40A/smo³ FRT40A ptc-lacZ; UAS-Dl/+</i>.</p