Mmot1, a New Helix-Loop-Helix Transcription Factor Gene Displaying a Sharp Expression Boundary in the Embryonic Mouse Brain

Several genetic factors have been proven to contribute to the specification of the metencephalic-mesencephalic territory, a process that sets the developmental foundation for prospective morphogenesis of the cerebellum and mesencephalon. However, evidence stemming from genetic and developmental studies performed in man and various model organisms suggests the contribution of many additional factors in determining the fine subdivision and differentiation of these central nervous system regions. In man, the cerebellar ataxias/aplasias represent a large and heterogeneous family of genetic disorders. Here, we describe the identification by differential screening and the characterization of Mmot1, a new gene encoding a DNA-binding protein strikingly similar to the helix-loop-helix factor Ebf/Olf1. Throughout midgestation embryogenesis, Mmot1is expressed at high levels in the metencephalon, mesencephalon, and sensory neurons of the nasal cavity. In vitro DNA binding data suggest some functional equivalence of Mmot1 and Ebf/Olf1, possibly accounting for the reported lack of olfactory or neural defects in Ebf −/− knockout mutants. The isolation of Mmot1 and of an additional homolog in the mouse genome defines a novel, phylogenetically conserved mammalian family of transcription factor genes of potential relevance in studies of neural development and its aberrations

MITO-Luc/GFP zebrafish model to assess spatial and temporal evolution of cell proliferation in vivo

Abstract We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo

Hypoxia/Reoxygenation Cardiac Injury and Regeneration in Zebrafish Adult Heart

<div><p>Aims</p><p>the adult zebrafish heart regenerates spontaneously after injury and has been used to study the mechanisms of cardiac repair. However, no zebrafish model is available that mimics ischemic injury in mammalian heart. We developed and characterized zebrafish cardiac injury induced by hypoxia/reoxygenation (H/R) and the regeneration that followed it.</p><p>Methods and Results</p><p>adult zebrafish were kept either in hypoxic (H) or normoxic control (C) water for 15 min; thereafter fishes were returned to C water. Within 2–6 hours (h) after reoxygenation there was evidence of cardiac oxidative stress by dihydroethidium fluorescence and protein nitrosylation, as well as of inflammation. We used Tg(cmlc2:nucDsRed) transgenic zebrafish to identify myocardial cell nuclei. Cardiomyocyte apoptosis and necrosis were evidenced by TUNEL and Acridine Orange (AO) staining, respectively; 18 h after H/R, 9.9±2.6% of myocardial cell nuclei were TUNEL⁺ and 15.0±2.5% were AO⁺. At the 30-day (d) time point myocardial cell death was back to baseline (n = 3 at each time point). We evaluated cardiomyocyte proliferation by Phospho Histone H3 (pHH3) or Proliferating Cell Nuclear Antigen (PCNA) expression. Cardiomyocyte proliferation was apparent 18–24 h after H/R, it achieved its peak 3–7d later, and was back to baseline at 30d. 7d after H/R 17.4±2.3% of all cardiomyocytes were pHH3⁺ and 7.4±0.6% were PCNA⁺ (n = 3 at each time point). Cardiac function was assessed by 2D-echocardiography and Ventricular Diastolic and Systolic Areas were used to compute Fractional Area Change (FAC). FAC decreased from 29.3±2.0% in normoxia to 16.4±1.8% at 18 h after H/R; one month later ventricular function was back to baseline (n = 12 at each time point).</p><p>Conclusions</p><p>zebrafish exposed to H/R exhibit evidence of cardiac oxidative stress and inflammation, myocardial cell death and proliferation. The initial decrease in ventricular function is followed by full recovery. This model more closely mimics reperfusion injury in mammals than other cardiac injury models.</p></div

Oxidative stress detection after H/R <i>in vivo.</i>

<p>DHE staining (a-d) and N-Tyr immunofluorescence (e-f) of hearts under control conditions (C) and exposed to H/R. (a) Representative confocal microscopy images of DHE staining in C and 2 h after H/R. (b) Merge of DHE and Hoechst nuclear staining. Calibration bar = 20 µm. White arrow-heads indicate DHE⁺ nuclei. (c) 3D representation of DHE fluorescence intensity distribution in the analyzed area: the <i>z-axis</i> shows the fluorescence intensity in cardiac nuclei, the <i>y-axis</i> and <i>x-axis</i> show the spatial distribution of nuclei on a plane. (d) Graph shows Mean Fluorescence Intensity (MFI) in C and 2 h to 14 h after H/R (n = 4 at each time point; ** <i>p</i><0.01 <i>vs.</i> C). Time course analysis revealed a peak of oxidative stress at 2 h in zebrafish adult heart sections, detected by DHE staining. (e) Representative confocal microscopy images of N-Tyr immunofluorescence, where green fluorescence indicates anti-N-Tyr and Hoechst nuclei staining: control (C, left panel) and 2 h after H/R (right panel). Calibration bar = 10µm. (f) Graph shows Mean Fluorescence Intensity (MFI) in C and 2 h to 14 h after H/R (n = 4 at each time point; ** <i>p</i><0.01 <i>vs.</i> C). H/R induced protein nitrosylation with a peak effect at the 2 h time point.</p

Detection of HIF-1α-dependent genes expression in whole hearts after H/R <i>in vivo.</i>

<p>Graphs show selected HIF-1α-dependent genes expression in whole hearts in control (C) and at different time points (3 h, 6 h, and 9 h) after H/R. (a) <i>Hmox1</i> mRNA expression exhibited a progressive increase and, at the 9 h time point, <i>hmox1</i> was ∼8-fold higher than in C. (b) <i>Vegfaa</i> mRNA increased and achieved its peak 6 h after H/R. (c) <i>Epo</i> mRNA exhibited a peak increase at 3 h which was ∼1.7-fold higher than in C but failed to achieve statistical significance. (n = 6; * <i>p</i><0.05 and *** <i>p</i><0.001 <i>vs.</i> C).</p

Inflammatory response induced by H/R <i>in vivo.</i>

<p>Representative confocal microscopy images (a-h) showing neutrophils (yellow fluorescence or green fluorescence) and macrophages (red fluorescence) infiltration in double transgenic line Tg(MPO:EGFP)×Tg(LysC:DsRed) in control (C) and at different time points (4 h, 6 h, and 14 h) after H/R. Neutrophils are either yellow (LysC⁺/MPO⁺) or green (predominantly, MPO⁺) cells (arrows in the 6 h image); red macrophages are LysC⁺ cells (arrow in the 4 h image). Hoechst stains cell nuclei; (a-d) calibration bar = 100 µm, (e-h) calibration bar = 20 µm.The peak inflammatory response occurred at the 6 h time point after H/R This experiment was performed three times with similar results.</p

Necrotic myocyte cell death induced by H/R <i>in vivo.</i>

<p>Necrotic myocyte cell death was assessed under baseline conditions, and 18 h and 30d after H/R in the Tg(cmlc2:nucDsRed) zebrafish line. At 18 h after H/R it was found a marked increase in necrotic myocardial cell number, which was back to control value at the 30d time point. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R showing colocalization of DAPI, DsRED and AO stainings. Arrows indicate cardiomyocyte AO⁺ nuclei. (b) AO⁺ cardiomyocytes nuclei in control (C) animals, and 18 h and 30d after H/R (n = 3 at each time point; ** <i>p</i><0.01 <i>vs.</i> C).</p

Apoptotic myocyte cell death induced by H/R <i>in vivo.</i>

<p>Apoptotic myocyte cell death was assessed under baseline conditions, and 18 h and 30d after H/R in the Tg(cmlc2:nucDsRed) zebrafish line. At 18 h after H/R it was found a marked increase in apoptotic myocardial cell number, which was back to control value at the 30d time point. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R, showing colocalization of DAPI, DsRED and TUNEL stainings. Arrows indicate cardiomyocyte TUNEL⁺ nuclei, whereas arrow-head indicates non-cardiomyocyte TUNEL⁺ nuclei. (b) TUNEL⁺ cardiomyocytes nuclei in control (C) animals, and 18 h and 30d after H/R (n = 3 at each time point; * <i>p</i><0.05 <i>vs.</i> C).</p

Myocardial cells positive for PCNA induced by H/R <i>in vivo.</i>

<p>Cardiomyocytes proliferation was assessed under baseline conditions and 18 h to 30d after H/R in Tg(cmlc2:nucDsRed) zebrafish line. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R showing colocalization of DAPI, DsRed and PCNA stainings. Arrows indicate cardiomyocyte PCNA⁺ nuclei. (b) Following H/R, there was a progressive increase in PCNA⁺ cardiomyocytes nuclei; the peak increase was achieved at the 3d time point, and at 30d the number of PCNA⁺ myocardial cells was back to control value (n = 3 at each time point; ** <i>p</i><0.01 and *** <i>p</i><0.001 <i>vs.</i> C).</p