Spire, an Actin Nucleation Factor, Regulates Cell Division during Drosophila Heart Development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis

Heart phenotypes in spir^2F mutants.

<p>(A–H) Compared to wild-type <i>Drosophila</i> embryos, <i>spir^2F</i> mutant embryos are characterized by paired nuclei, larger cells and fewer cardioblasts at stage 16. (A, B) The <i>Toll-GFP</i> transgene expresses GFP in cardioblasts. (C, D) The <i>Hand-GFP</i> transgene and (E, F) the <i>Tup-GFP</i> transgene expresses GFP in cardioblasts, pericardial cells and the lymph gland. (G, H) Spectrin (green) and Mef2 (red) stain membrane skeleton and cardioblasts, respectively. (I, J) At stage 12, Eve stains pericardial cells and DA1 cells. In <i>spir^2F</i> mutant embryos, Eve-positive cells appear in pairs. All embryos are oriented with the anterior to the left. All the images are taken under 40× magnification. Arrows indicate paired nuclei in <i>spir</i> mutants. Pericardial cells (PC), cardioblasts (CB), Eve-positive pericardial cells (EPC) and Eve-positive DA1 cells (EDA1) of the normal dorsal vessel are indicated in the wild-type embryos.</p

A cell division model of <i>spir</i> function during heart development.

<p>(A) In wild-type embryos, there are six cardioblasts in each hemi-segment including two Svp-positive and four Tin-positive cells. An SSP divides into two SPs, then each of the SPs divides into one SC and one SPC. After two cell cycles, one TSP differentiates into 4 TCs. (B) In <i>CycA</i> mutants, cell cycle 16 is blocked so that there are two SCs and two TCs in each hemi-segment. However, there are no SPCs. (C) In <i>spir</i> mutants, the nucleus duplicates and divides while the cytoplasm does not. Most of the SSPs stop division at cell cycle 15 resulting in double-nucleated cells. Some of the SSPs divide into two SPs, but stop at cell cycle 16 giving rise to four or three nuclei clusters. TPs are arrested at cell cycle 16 leading to two double-nucleated cells in each hemi-segment. SSP, Svp-positive super-progenitor; TSP, Tin-positive super-progenitor; SP, Svp-positive progenitor; TP, Tin-positive progenitor; SC, Svp-positive cardioblasts; TC, Tin-positive cardioblast; SPC, Svp-positive pericardial cells. The dotted circle represents the cell membrane.</p

Pattern of Tin- and Svp-positive cells in <i>wt</i>, <i>CycA^C8LR1</i> and <i>spir^2F</i> embryos.

<p>(A, A′, B and B′) Lateral view at stage 13. (C, C′, D and D′) Dorsal view at stage 15. (E–H) Dorsal view at stage 16. Mef2 (red) stains Mef2-positive cardioblasts and β-Gal (green) stains Svp-positive cardioblasts. In <i>spir^2F</i> mutants, when two nuclei appear together, most cells express only Svp. When three or four nuclei appear together, they are stained with both anti- β-Gal and anti-Mef2. SC, Svp-positive cardioblasts; TC, Tin-positive cardioblasts. Arrows point to Svp-positive cardioblasts; Arrowheads point to Svp-positive pericardial cells. * indicates Svp-positive cells; — indicates Tin-positive cells.</p

Genetic interactions between <i>spir</i> and <i>Doc</i>, <i>tin</i>, <i>pnr</i>.

<p>(A) Wild-type embryo. (B–D) Double heterozyous embryos of <i>spir</i> and <i>Df(3L)DocA</i>, <i>spir</i> and <i>tin</i> and <i>spir</i> and <i>pnr</i>, respectively. Embryos are all at stage 16. The mutant phenotypes of all three double heterozygous embryos show missing cardioblasts. (E–H) Spir antibody staining in <i>wt</i> and <i>pnr</i> mutant, <i>wt</i> and <i>tin</i> mutant embryos, respectively. (E, F) In <i>pnr</i> mutant embryos, Spir is over-expressed in the cardiac mesoderm, compared to the ubiquitous expression pattern in wild-type embryo at stage 13. (G, H) There is no Spir expression in heart cells in <i>tin</i> mutants at stage 16. The cells that express <i>Toll-GFP</i> and Spir are amnioserosa cells. (I, J) Tin antibody staining in wild-type embryo (I) and <i>spir</i> mutant embryo (J) at stage 13. Tin cells appear in pairs and are disorganized. Arrows highlight the abnormal positioning of cardioblasts in different mutant embryos. ★ indicates cardiac mesoderm.</p

Genetic interaction between <i>spir</i> and <i>CycA</i>.

<p>This interaction results in various phenotypes. (A, A′, B and B′) Stage 13. In each hemi-segment, there are two Tin-positive cells and one Svp-positive cell in <i>spir</i> and <i>CycA</i> double heterozygous embryos (B and B′). (C–C″ and D–D″) Stage 16. Many cells are missing in the aorta region (D–D″). Many mutant embryos show a phenotype between these two extremes. Arrows emphasize cellular gaps in the dorsal vessel. * indicates Svp-positive cells; — indicates Tin-positive cells. Arrowheads point to paired nuclei. Arrows point to the missing cells.</p

Statistical analysis of genetic interaction between <i>spir</i> and <i>tin</i>, <i>spir</i> and <i>Doc</i>, <i>spir</i> and <i>pnr</i>, <i>spir</i> and <i>CycA</i>.

<p>**The χ² test reveals in all three groups that the proportion of mutant and normal embryos is statistically different for single heterozygous and double heterozygous embryos.</p><p>*The χ² statistic is not significant at the 0.05 level, but at the 0.1 level.</p>#<p>Mutant hearts in these double heterozygotes denote gap phenotype.</p

Screening and Analysis of Janelia FlyLight Project Enhancer-Gal4 Strains Identifies Multiple Gene Enhancers Active During Hematopoiesis in Normal and Wasp-Challenged Drosophila Larvae

A GFP expression screen has been conducted on >1000 Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes, with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to our knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process