Diminished Telomeric 3′ Overhangs Are Associated with Telomere Dysfunction in Hoyeraal-Hreidarsson Syndrome

BACKGROUND:Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency. METHODOLOGY/PRINCIPAL FINDINGS:We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3' overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types. CONCLUSIONS/SIGNIFICANCE:Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease

Novel interplay between JNK and Egfr signaling in <i>Drosophila</i> dorsal closure

<div><p>Dorsal closure (DC) is a developmental process in which two contralateral epithelial sheets migrate to seal a large hole in the dorsal ectoderm of the <i>Drosophila</i> embryo. Two signaling pathways act sequentially to orchestrate this dynamic morphogenetic process. First, c-Jun N-terminal kinase (JNK) signaling activity in the dorsal-most leading edge (LE) cells of the epidermis induces expression of <i>decapentaplegic</i> (<i>dpp</i>). Second, Dpp, a secreted TGF-β homolog, triggers cell shape changes in the adjacent, ventrally located lateral epidermis, that guide the morphogenetic movements and cell migration mandatory for DC. Here we uncover a cell non-autonomous requirement for the Epidermal growth factor receptor (Egfr) pathway in the lateral epidermis for sustained <i>dpp</i> expression in the LE. Specifically, we demonstrate that Egfr pathway activity in the lateral epidermis prevents expression of the gene <i>scarface</i> (<i>scaf</i>), encoding a secreted antagonist of JNK signaling. In embryos with compromised Egfr signaling, upregulated Scaf causes reduction of JNK activity in LE cells, thereby impeding completion of DC. Our results identify a new developmental role for Egfr signaling in regulating epithelial plasticity via crosstalk with the JNK pathway.</p></div

Egfr signaling is positively required for the full expression of the JNK pathway target gene <i>dpp</i> and for Mad phosphorylation.

<p>(A-F) Lateral (A, B, D, E) or dorsolateral (C, F) views of embryos hybridized using a digoxigenin-labeled RNA probe for <i>dpp</i> (blue). (A’-F’) Corresponding magnified views of the regions marked by black arrowheads in panels (A-F). (A”-F”) show embryos stained for pMad (red). (A-A”) Wild-type embryo showing the normal <i>dpp</i> (A-A’) and pMad (A”) patterns. Levels of <i>dpp</i> and pMad are reduced in <i>rhomboid</i> mutants (D-D”), as well as in embryo expressing <i>pnr>Egfr</i>^<i>DN</i> (E- E”). Conversely, both expand in embryo expressing <i>pnr>Ras</i>^<i>V12</i> (F- F”). These effects largely phenocopy loss- or gain-of-function JNK signaling (<i>bsk</i> mutant and <i>pnr>Hep</i>^<i>Act</i> embryo in B-B” and C-C”, respectively).</p

Dorsal closure is defective in embryos deficient for Egfr signaling.

<p>(A-H) Cuticle preparations. (A) Wild-type cuticle; note the complete closure of the epidermis on the dorsal side. (B) <i>bsk</i> mutant embryo; note the characteristic dorsal-open phenotype (arrowhead). (C-H) Lack of functional Egfr signaling leads to the formation of dorsal-open holes (arrowheads), a phenotype typically associated with JNK pathway mutants (cf. B). Egfr pathway activity was compromised by <i>pnr>Gal4</i>-driven ectodermal expression of <i>Egfr</i>^<i>DN</i> (C) or <i>Ras</i>^<i>DN</i> (D), or in <i>rhomboid</i> (E), <i>spi</i> (F) and allelic <i>Egfr</i> (G-H) mutant embryos.</p

Over-expression of <i>scarface</i> mimics the loss of Egfr pathway activity.

<p>(A-B) Embryos hybridized using a digoxigenin-labeled RNA probe for <i>dpp</i> (blue). Over-expression of <i>scaf</i> brings about a reduction in <i>dpp</i> expression (A) whereas <i>dpp</i> expression expands in <i>scaf</i> mutant embryos (B), similarly to loss- and gain-of-function mutations in the Egfr pathway, respectively. (C-F) Embryos stained for pMad (red). (C, D) In keeping with <i>dpp</i> expression levels, pMad staining decreases upon <i>scaf</i> over-expression (C), and is augmented in a <i>scaf</i> mutant (D). (E, F) Although pMad staining is reduced in <i>rhomboid</i> single mutant embryo (E), it expands in embryo doubly mutant for <i>scaf</i> and <i>rhomboid</i> (F), as in <i>scaf</i> single mutant (D), indicating that <i>scaf</i> is epistatic to <i>Egfr</i> signaling. (G-H) Model showing how Egfr signaling in the lateral epidermis positively and non-autonomously contributes to JNK pathway activity in LE cells and to DC. (G) The Egfr pathway normally acts in the lateral epidermis to prevent expression of the JNK antagonist, <i>scaf</i>, thus supporting maximal JNK activity in LE cells. (H) When Egfr signaling is defective, deregulated Scaf subsequently attenuates functional JNK signaling in LE cells, thus hindering the process of DC. Bold text and arrows/bars indicate normal levels of gene expression and regulation, whereas gray fonts designate abnormally lower levels of expression and regulation, respectively.</p

The Egfr pathway induces expression of Engrailed, a <i>scarface</i> repressor, in the lateral epidermis.

<p>(A, A’) Cuticle preparation. Dark (A) and bright field (A’) images of an embryo expressing <i>pnr>Yan</i>^<i>Act</i>. Note the dorsal open hole. (B, C) Embryos expressing <i>pnr>Yan</i>^<i>Act</i>, hybridized using a digoxigenin-labeled RNA probe for <i>dpp</i> (blue; B) or stained for pMad (red; C). (B’) Magnified view of the region marked by a rectangle in (B). Note that Yan^Act brings about a reduction in <i>dpp</i> expression and, as a consequence, a reduction in the pMad domain, similarly to other <i>Egfr</i> pathway mutants. (D) Embryo expressing <i>pnr>Yan</i>^<i>Act</i> hybridized using a digoxigenin-labeled RNA probe for <i>scaf</i> (blue). (D’) Magnified view of the region marked by a rectangle in (D). Note that <i>scaf</i> expression expands into the lateral epidermis. (E, F) Yan^ACT dominantly represses En. Control embryo expressing <i>pnr>GFP</i> (E) and embryo expressing <i>pnr>Yan</i>^<i>Act</i> (F) stained for En (green), as well as for LacZ (magenta; <i>puc-lacZ</i>) to mark the LE. Yan^ACT activity reduces En expression in the LacZ-positive LE cells, as well as in the adjacent lateral epidermis (F). (G) Model explaining how Egfr signaling prevents expression of <i>scaf</i> in the lateral epidermis.</p

Egfr signaling acts upstream of the JNK cascade in dorsal closure.

<p>(A-F) Embryos stained for pMad (red). pMad staining decreases in embryos expressing <i>pnr>Ras</i>^<i>DN</i> (A) and expands in embryo expressing <i>pnr>Hep</i>^<i>Act</i> (B). In embryos co-expressing <i>pnr>Hep</i>^<i>Act</i>; <i>Ras</i>^<i>DN</i>, pMad staining expands (C). pMad staining expands in embryo expressing <i>pnr>Ras</i>^<i>V12</i> (D) and is largely reduced in embryo expressing <i>pnr>Bsk</i>^<i>DN</i> (E). In embryos co-expressing <i>pnr>Bsk</i>^<i>DN</i><i>; Ras</i>^<i>V12</i>, pMad staining is also reduced (F). These results indicate that JNK signaling is epistatic to the Egfr pathway.</p

EGFR signaling takes place in the lateral epidermis of stage 13 embryos, at the time of dorsal closure.

<p>(A) A schematic lateral view of a stage 13 embryo (st13; 9:20–10:20 hours after egg lay). Demarcated are three groups of cells that participate in the process of DC: the amnioserosa (yellow), the dorsal-most row of ectodermal cells termed leading edge cells (green) and the adjacent lateral epidermis cells (red). (B-D) A <i>rhomboid</i>-<i>lacZ</i> enhancer-trap embryo co-stained for dpErk (B; red) and LacZ (C; green). (D) Merge. (B, D) dpErk staining is evident in the lateral epidermis. (C, D) dpErk staining borders on <i>lacZ</i> expression. (E-I) dpErk staining (red) is greatly reduced in the lateral epidermis of embryos expressing <i>pnr>Egfr</i>^<i>DN</i> (E), or in embryos mutant for <i>rhomboid</i> (F), <i>spi</i> (G) and <i>Egfr</i> (H), though not in <i>pvr</i> mutants (I). In (E) and (H), the signal in the AS is an artifact caused by auto-florescence. In this and all other Figures, embryos are at st13 and presented in lateral views, with anterior to the left and dorsal up, unless otherwise stated.</p

Functional Egfr signaling is required for the cell shape changes that occur during dorsal closure.

<p>(A-F) Embryos stained for DE-cadherin (green) to outline cell membranes. Corresponding primed panels (A’-F’) show magnified views of the regions marked with arrowheads. (A) Wild-type embryo. At this stage, both LE cells and cells in the lateral epidermis elongate and stretch along the D/V axis. (B) Elongation failure typifies <i>bsk</i> and other mutant embryos defective in JNK pathway signaling. (C-F) Deficiency in Egfr signaling in embryos mutant for <i>spi</i> (C) or <i>rhomboid</i> (D), as well as in those expressing <i>pnr>Egfr</i>^<i>DN</i> (E) or <i>pnr>Ras</i>^<i>DN</i> (F), leads to failure of epidermal cells to elongate. Instead, they remain polygonal, thus phenocopying <i>bsk</i> mutants (B).</p