HEXIM knockdown affects both differentiating and proliferating tissues.

<p>(A) Schematic diagram of the eye-antenna disc summarizing the expression patterns of the <i>Gal4</i> drivers: <i>ey</i> (red), <i>GMR</i> (green) and <i>so</i> (hatched). (B) WT and <i>GMR-Gal4>RNAi Hexim</i> eyes. (C) WT and <i>so-Gal4>RNAi Hexim</i> eyes. (D) WT and the <i>ey-Gal4</i>><i>RNAi Hexim e</i>ye-antenna imaginal discs (white circles) and brain. Note the absence of eye-antenna discs in <i>ey-Gal4</i>><i>RNAi Hexim</i> (white arrows).</p

HEXIM knockdown deregulates Ci¹⁵⁵ expression at both protein and transcript levels.

<p>Expression of Ci¹⁵⁵ and En in <i>rn-Gal4>RNAi Hexim</i> (A) and WT (B) wing discs, at various L3 stages. (C) Transcription of the <i>Ci-lacZ</i> reporter in WT and <i>rn-Gal4>RNAi Hexim</i> wing discs.</p

Apoptosis is reduced in double RNAi-mediated knockdown mutant of HEXIM and Ci or HEXIM and Hh.

<p>Immunodetection of cleaved caspase 3 (Casp3*) and Ci¹⁵⁵ at early L3 stage of WT (A), <i>rn-Gal4>RNAi Hexim</i> (B), <i>rn-Gal4>RNAi Ci</i> (C), <i>rn-Gal4>RNAi Hexim; RNAi Ci</i> (D), <i>rn-Gal4>RNAi Hh</i> (E) and <i>rn-Gal4>RNAi Hexim; RNAi Hh</i> (F) strains.</p

Reduction of Hh does not rescue HEXIM knockdown mutant.

<p>(A) Wing phenotype in WT and <i>rn-Gal4>RNAi Hh</i> flies. The distance between L3 and L4 veins are indicated with a red bar. Immuno-localization of Ci¹⁵⁵ and Ptc in WT (B), <i>rn-Gal4>RNAi Hh</i> (C) and <i>rn-Gal4>RNAi Hexim; RNAi Hh</i> double mutant (C) wing discs. The reduced levels of Ptc in the A-P stripe (white arrow) are marked in single and double mutants.</p

Model of HEXIM-dependent regulation of wing disc development.

<p>(A) In WT wing pouch, HEXIM regulates hedgehog signaling and so its transcriptional effector Ci. (B) In HEXIM knockdown background, Hh is strongly induced and so Ci, and provokes apoptosis, which activates apoptosis-mediated compensatory proliferation. The resulting patterning defects prevent wing development despite the compensatory proliferation.</p

Co-expression of p35 or dMyc/Cyclin E partially rescue HEXIM knockdown phenotype.

<p>(A) Adult wing, (B) DAPI stained wing disc and (C) Phospho-Histone 3 (P-H3) immunodetection at early L3 stage of WT, <i>rn-Gal4>UAS-p35</i>, <i>rn-Gal4>RNAi Hexim</i> and <i>rn-Gal4>UAS-p35</i>; <i>RNAi Hexim</i> flies.</p

HEXIM knockdown deregulates Hh signaling pathway.

<p>(A) X-Gal staining of <i>hh-lacZ</i> reporter, in WT and <i>rn-Gal4>RNAi Hexim</i> wing discs. For the mutant, β-galactosidase staining duration was reduced to limit signal saturation. En and Hh-LacZ β-galactosidase co-immunodectection in WT (B) and in <i>rn-Gal4>RNAi Hexim</i> (C) wing discs. Arrows indicate the non-autonomous down-regulation of Hh in the posterior compartment of the notum part. The wing pouch is marked with dotted white line. The assays were performed at early L3 stage.</p

HEXIM knockdown induces cell death and transient systemic proliferation arrest.

<p>(A) Expression of HEXIM in WT and <i>rn-Gal4>RNAi Hexim</i> wing discs at early L3 stage. (B) Immunodetection of cleaved caspase 3 (Casp3*) and (C) Phospho-Histone 3 (P-H3) in WT and <i>rn-Gal4>RNAi Hexim</i> wing discs at early L3 stage. The scale bar is for 100μm. In this and all subsequent figures, wing discs are orientated anterior (an) at left and posterior (po) at right.</p

Table_7_Overlapping action of T3 and T4 during Xenopus laevis development.xlsx

Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter thyroid hormone signaling and induce unwanted effects on human and ecosystems health. Regulatory tests have been developed to detect these compounds but need to be significantly improved by proposing novel endpoints and key events. The Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on Xenopus laevis tadpoles, a particularly sensitive model system for studying the physiology and disruption of thyroid hormone signaling: amphibian metamorphosis is a spectacular (thus easy to monitor) life cycle transition governed by thyroid hormones. With a long-term objective of providing novel molecular markers under XETA settings, we propose first to describe the differential effects of thyroid hormones on gene expression, which, surprisingly, are not known. After thyroid hormones exposure (T3 or T4), whole tadpole RNAs were subjected to transcriptomic analysis. By using standard approaches coupled to system biology, we found similar effects of the two thyroid hormones. They impact the cell cycle and promote the expression of genes involves in cell proliferation. At the level of the whole tadpole, the immune system is also a prime target of thyroid hormone action.</p

Table_4_Overlapping action of T3 and T4 during Xenopus laevis development.xlsx

Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter thyroid hormone signaling and induce unwanted effects on human and ecosystems health. Regulatory tests have been developed to detect these compounds but need to be significantly improved by proposing novel endpoints and key events. The Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on Xenopus laevis tadpoles, a particularly sensitive model system for studying the physiology and disruption of thyroid hormone signaling: amphibian metamorphosis is a spectacular (thus easy to monitor) life cycle transition governed by thyroid hormones. With a long-term objective of providing novel molecular markers under XETA settings, we propose first to describe the differential effects of thyroid hormones on gene expression, which, surprisingly, are not known. After thyroid hormones exposure (T3 or T4), whole tadpole RNAs were subjected to transcriptomic analysis. By using standard approaches coupled to system biology, we found similar effects of the two thyroid hormones. They impact the cell cycle and promote the expression of genes involves in cell proliferation. At the level of the whole tadpole, the immune system is also a prime target of thyroid hormone action.</p