Functional Interaction between HEXIM and Hedgehog Signaling during Drosophila Wing Development

International audienceStudying the dynamic of gene regulatory networks is essential in order to understand the specific signals and factors that govern cell proliferation and differentiation during development. This also has direct implication in human health and cancer biology. The general transcrip-tional elongation regulator P-TEFb regulates the transcriptional status of many developmental genes. Its biological activity is controlled by an inhibitory complex composed of HEXIM and the 7SK snRNA. Here, we examine the function of HEXIM during Drosophila development. Our key finding is that HEXIM affects the Hedgehog signaling pathway. HEXIM knockdown flies display strong phenotypes and organ failures. In the wing imaginal disc, HEXIM knock-down initially induces ectopic expression of Hedgehog (Hh) and its transcriptional effector Cubitus interuptus (Ci). In turn, deregulated Hedgehog signaling provokes apoptosis, which is continuously compensated by apoptosis-induced cell proliferation. Thus, the HEXIM knock-down mutant phenotype does not result from the apoptotic ablation of imaginal disc; but rather from the failure of dividing cells to commit to a proper developmental program due to Hedgehog signaling defects. Furthermore, we show that ci is a genetic suppressor of hexim. Thus, HEXIM ensures the integrity of Hedgehog signaling in wing imaginal disc, by a yet unknown mechanism. To our knowledge, this is the first time that the physiological function of HEXIM has been addressed in such details in vivo

Perinatal and early postnatal changes in the expression of monocarboxylate transporters MCT1 and MCT2 in the rat forebrain

In addition to glucose, monocarboxylates including lactate represent a major source of energy for the brain, especially during development. We studied the immunocytochemical expression of the monocarboxylate transporters MCT1 and MCT2 in the rat brain between embryonic day (E) 16 and postnatal day (P) 14. At E16-18, MCT1-like immunoreactivity was found throughout the cortical anlage, being particularly marked medially in the hippocampal anlage next to the ventricle. In a complementary pattern, MCT2-like immunoreactivity was expressed along the medial and ventral border of the ventricle in the medial septum and habenula before birth. The hypothalamic area exhibited MCT2 and MCT1 positive areas from E18 on. These transient labelings revealed four main sites of monocarboxylate and/or glucose exchange: the brain parenchyma, the epithelial cells, the ependymocytes, and the glia limitans. During the first postnatal week, MCT1 immunoreactivity extended massively to the vessel walls and moderately to the developing astrocytes in the cortex. In contrast, MCT2 immunoreactivity was faint in blood vessels but massive in developing astrocytes from P3 to P7. Neither MCT2 nor MCT1 colocalized with neuronal, microglial, or oligodendrocytic markers during the first postnatal week. At P14, a part of the scattered punctate MCT2 staining could be associated with astrocytes and postsynaptic dendritic labeling. The transient pattern of expression of MCTs throughout the perinatal period suggests a potential relationship with the maturation of the blood-brain barrier

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

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

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

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

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