A Novel Role for Micos Complex CHCHD3/6 in Cardiac Function and Structure

Presentation of poster 2079C at TACG 2020 online. Files include a PDF of the poster (Birker, Katja_TAGC2020.pdf) and a 10 minute video walkthrough of the poster material (Birker, Katja_TAGC2020.m4a).</div

Elucidating the Role of Uncharacterized Tin-Positive Pericardial Cells in Drosophila Heart Development

Drosophila is an excellent model system for studying developmental biology because of its small size, short generation time, the abundance of eggs laid, and the plethora of genetic tools for the manipulation of gene expression in time and space. In addition, many genes that cause human heart diseases are found in Drosophila and play similar roles in heart function and development. We are studying heart development at high spatio-temporal resolution in the fruit fly by single cell RNA sequencing of cardiac cells and whole mount in situ hybridization. Specifically, we are focusing on novel genes involved in heart development in fly embryos to learn what stages of development these genes are expressed and, using mutational analysis, to what extent they are required for the formation and differentiation of cardiac tissue.We have found new marker genes for a group of uncharacterized embryonic pericardial cells that express the cardiac master regulator Tinman (tin-positive pericardial cells, TPCs), but not Even- or Odd-skipped, the other markers for pericardial cells. These TPCs co-express a specific set of genes, including cut, Wnt4, Nrt, Scb, CrzR, and DOR. We are conducting immunofluorescent staining and imaging of fly embryos to visualize the protein and mRNA expression patterns of these marker genes in the entire embryo, the heart, and heart-associated tissues. In addition, we are characterizing these cells to learn about their cell-specific roles during heart development. For the purpose of studying these roles in heart development, we are knocking down or overexpressing them in the embryonic heart using heart-specific Gal4 driver lines and examining trans-heterozygous combinations of loss of function alleles for these genes.By determining the gene regulatory program of the TPCs and characterizing in-depth the development and function of these pericardial cells, we hope to elucidate their role in Drosophila heart development.</p

Multiplexin Promotes Heart but Not Aorta Morphogenesis by Polarized Enhancement of Slit/Robo Activity at the Heart Lumen

<div><p>The <i>Drosophila</i> heart tube represents a structure that similarly to vertebrates' primary heart tube exhibits a large lumen; the mechanisms promoting heart tube morphology in both <i>Drosophila</i> and vertebrates are poorly understood. We identified Multiplexin (Mp), the <i>Drosophila</i> orthologue of mammalian Collagen-XV/XVIII, and the only structural heart-specific protein described so far in <i>Drosophila</i>, as necessary and sufficient for shaping the heart tube lumen, but not that of the aorta. Mp is expressed specifically at the stage of heart tube closure, in a polarized fashion, uniquely along the cardioblasts luminal membrane, and its absence results in an extremely small heart tube lumen. Importantly, Mp forms a protein complex with Slit, and interacts genetically with both <i>slit</i> and <i>robo</i> in the formation of the heart tube. Overexpression of Mp in cardioblasts promotes a large heart lumen in a Slit-dependent manner. Moreover, Mp alters Slit distribution, and promotes the formation of multiple Slit endocytic vesicles, similarly to the effect of overexpression of Robo in these cells. Our data are consistent with Mp-dependent enhancement of Slit/Robo activity and signaling, presumably by affecting Slit protein stabilization, specifically at the lumen side of the heart tube. This activity results with a Slit-dependent, local reduction of F-actin levels at the heart luminal membrane, necessary for forming the large heart tube lumen. Consequently, lack of Mp results in decreased diastolic capacity, leading to reduced heart contractility, as measured in live fly hearts. In summary, these findings show that the polarized localization of Mp controls the direction, timing, and presumably the extent of Slit/Robo activity and signaling at the luminal membrane of the heart cardioblasts. This regulation is essential for the morphogenetic changes that sculpt the heart tube in <i>Drosophila</i>, and possibly in forming the vertebrates primary heart tube.</p></div

A scheme describing the molecular interactions between Mp, Slit/Robo and F-actin in the heart and the aorta.

<p>A- Heart-specific expression of Mp that is restricted to the luminal membrane, promotes the formation of Mp-Slit complex facilitating Slit/Robo activity, leading to the formation of Slit endocytic vesicles close to the luminal membrane. Slit/Robo signaling reduces F-actin levels at the luminal membrane, and restricts the junctional domain by promoting Armadilo turn over. Reiterated Slit/Robo signaling is maintained as a result of continued Mp expression and its association with newly secreted Slit, leading to inward curving of the luminal membrane. B- Lack of Mp in the aorta limits Slit/Robo activation, leading to a significantly narrow lumen.</p

Mp activity in cardioblasts depends on Slit.

<p>Cardiac cross sections of wild type (A, A′, A″), <i>slit</i> mutants (B, B′, B″), embryos overexpressing Mp in cardioblasts (C, C′,C″), embryos overexpressing Robo in cardioblasts (D, D′,D″), and embryos overexpressing Mp in <i>slit</i> mutant background (E, E′, E″), all labeled with anti Dg (red) and with anti Armadillo (green). Arrowheads in A–A″ mark the dorsal and ventral junctions and in B–E″ the dorsal junction. Note that overexpression of Mp is not capable of promoting large and curved lumen in <i>slit</i> mutant hearts, and the lack of Armadillo vesicles in these cells. C′ and D′- White arrows mark Armadillo vesicles. C-cardioblast cells. The bar in A is 5 µm and represents the magnification in all panels.</p

Mp exhibits heart lumen-specific distribution and is necessary and sufficient for cardiac lumen formation.

<p>Upper panel: a scheme of the three stages of cardiac tube closure: I- cardioblasts approach the dorsal midline. II- formation of the dorsal junction and the inward curvature of the luminal membrane. III- formation of the ventral junction and tube closure. Mp initial expression is observed between stage II to stage III (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003597#pgen.1003597.s001" target="_blank">Figure S1</a>). A whole mount wild type embryo at stage 16 labeled for Mp (A,A′, green), and Mef-2 (A, red). The heart and aorta domains are indicated. The arrowhead in A indicates a pair of ostia cells which do not express Mp. B-I are cross sections of stage 16 embryos. Wild type heart (B,B′) or aorta (C,C′) labeled with Dystroglycan (Dg, red, B,C) and Mp (green B,B′,C,C′) demonstrate the cardiac specific distribution of Mp in the heart lumen. D-I are cross sections of: wild type heart (D), <i>mp</i> mutant heart (E), heart cardioblasts overexpressing Mp (F), wild type aorta (G) <i>mp</i> mutant aorta (H), aorta cardioblasts overexpressing Mp (I), labeled with Dg (red) and with anti HOW, which labels the cardioblast cytoplasm (green). Arrowheads in D-I mark the cardiac lumen. Note the formation of large cardiac lumen (29 µm², F), following Mp overexpression in the heart and the formation of a heart-like lumen in the aorta following Mp overexpression in the aorta. J - quantification of the lumen cross section area, K- quantification of the luminal perimeter, L- quantification of cardioblast cross section area measured from 3–4 cross sections per embryo in multiple number of embryos (n). A statistically significant reduction (indicated by three stars) in both cardiac luminal area (reduction of 55%, p = 8.6E-0.6) and perimeter (reduction of 27%, p = 3.8E-0.50) was observed. A slight reduction in cardioblasts total area was also detected (13%, p = 0.02, one star). C- cardioblast cell. Scale bars represent 20 µm in A,A′, and 5 µm in all cross sections (B,C, D–L).</p

Mp activity reduces F-actin levels at the cardiac luminal membrane in a <i>slit</i> dependent manner.

<p>Cardiac cross sections of embryos at stage 15 (A, A′) or stage 16 (B–G′) labeled with phalloidin (green) and Dg (red), of the following genotypes: wild type (A, A′, B, B′), <i>slit</i> mutant (C, C′), <i>mp</i> mutant (D, D′), embryos overexpressing Mp (<i>mef2-GAL4>UAS-mp</i>, E, E′), embryos overexpressing Robo (<i>mef-2-GAL4>UAS-robo</i>, F, F′) or <i>slit</i> mutant embryos overexpressing Mp (G, G′). White arrows indicate the luminal membrane, while the luminal membrane of stage 16 wild type and Mp overexpressing embryos displays reduced F-actin levels, the luminal membrane of <i>slit</i> and <i>mp</i> mutant exhibits elevated F-actin levels. Note that overexpression of Mp in <i>slit</i> mutant background did not reduce F-actin levels at the luminal membrane although a small lumen was detected. The scheme in H summarizes the results; in wild type (WT) heart a constitutive activation of Slit/Robo at the luminal membrane, promoted by Mp reduces F-actin levels at the luminal membrane. In <i>mp</i> mutants Slit/Robo signaling is reduced, and consequently F-actin levels are elevated leading to a small lumen. In contrast, overexpression of Mp (Mp OE) leads to elevated Slit/Robo signaling, reducing luminal F-actin levels and enhancing lumen size. Mp overexpression in the absence of Slit exhibited elevated levels of luminal F-actin and a small lumen. Bar in A is 5 µm and represents magnification of all panels.</p

Mp activity is essential for proper heart contraction of adult fly hearts.

<p>Cardiac cross sections through an adult heart stained with anti Mp (red, A,A′) and anti Laminin (green, A′). Empty white arrowheads in A and A′ indicate positive staining for Mp in the cardiac lumen. Filled white arrowheads show positive staining of Mp between the cardioblast layer and the ventral longitudinal muscles. B- Heart contractility is quantified as fractional shortening or the extent to which the heart walls come together during systole. Fractional shortening is significantly reduced in <i>mp</i> mutant flies. C- the reduction in fractional shortening is also evident in representative Mmode records from wild type (WT) or <i>mp</i> mutant hearts. Diastole and systole levels are indicated by red arrows.</p

Mp enhances Slit/Robo activity in the heart lumen and modulates Slit distribution in the central nervous system.

<p>Cross sections through the heart (A–F′″) or the aorta (G–H′) of wild type embryos (A, A′, G, G′); <i>robo</i> mutant (B, B′), embryos overexpressing Robo in cardioblasts (C, C′); <i>mp</i> mutant (D, D′), or embryos overexpressing Mp in cardioblasts (E, E′, H, H′) labeled with anti Slit (red) and anti Dg (green). F–F′″ show embryos expressing dominant-negative Rab5-YFP in cardioblasts labeled with Slit (F, red), YFP (F′, green), Slit and YFP (F″), or Slit and Dg (white, F′″). Panels A–H′ are single optical confocal sections to enable comparison of the extent of cytoplasmic Slit vesicles in each genotype. Insets are 2.5 folds enlargement of the heart luminal domain. White arrows in each inset, indicate Slit vesicle/s, with the exception of E′″ where the white arrow indicates Slit vesicle position. Note the reduction in the number and size of Slit endocytic vesicles observed in both <i>mp</i> and robo mutants, and their elevation following Mp and Robo overexpression. Represented schemes of Slit vesicles distribution are indicated for each genotype. Note the alteration in Slit distribution following Rab5 dominant negative overexpression (the majority of the protein overlaps Dg at the luminal membrane, and the cytoplasmic Slit is associated with the luminal membrane). I and I′ show wild type (I), or embryo overexpressing Mp in the midline using <i>sim-GAL4</i> driver (I′), both labeled for Fasciclin II (red). Note the shortening of the distance between the longitudinal commissures and the midline (marked by the white arrow), following overexpression of Mp. Cross sections through the ventral nerve cord of wild type (J,J′) or an embryo overexpressing Mp in the midline cells (K,K′) labeled with anti Slit (red) and with anti HOW (which marks the midline glia, green J′,K′). High Slit accumulation in the midline (marked by white arrowheads) is observed following overexpression of Mp. Bar in A is 5 µm and represents the magnification in panels A–H′. Bar in I is 20 µm, and represents magnification in I,I′. Bar in J is 10 µm and represents magnification in J–K′).</p

Genetic and physical association between Mp, Slit and Robo.

<p>Cardiac cross sections of wild type (A), <i>slit/+;mp/+</i> (B), <i>robo/+;mp/+</i> (C) double heterozygous, and <i>mp/+</i> (D), <i>slit/+</i> (E), <i>robo/+</i> (F) single heterozygous labeled with anti HOW (green), and anti Dg (red). The cardiac lumens (marked by arrowheads) of the double-heterozygous mutant are smaller relative to the control. G-I: cardiac cross sections of wild type embryos labeled with anti Slit (G,I red) and anti Mp (H,I green), indicating their co-localization along the lumen. J- immunoprecipitation with anti Slit antibodies (or with a control normal mouse serum) of an extract of S2 cells co-transfected with Slit, Robo, and Mp. The same blot was then reacted individually with anti- Slit, Mp, and Robo corresponding to the three upper lanes). The anti Mp antibody reacted with a single band of ∼120 kDa, corresponding to mp cDNA 3hnc1. This immunoprecipitation (IP) is representative of three independent IP experiments. The crude extracts contained comparable amounts of transfected proteins as indicated by the antibody reactivity with each of the transfected cDNA constructs presented in the right panel. K-Immunoprecipitation with anti Slit antibodies of comparable protein extracts taken from stage 16 control (yw), <i>mp^−/−</i>, or embryos overexpressing Mp in heart and muscles (using <i>mef2-GAL4</i> driver). Western blot with anti Slit of the IP material shows elevated levels of Slit in the Mp-overexpressing embryos and reduced Slit levels in <i>mp</i> mutants. Reaction of the same blot with anti Mp antibodies (lower panel) revealed a specific band of ∼39 kDa, corresponding to the Endostatin fragment. Western blot with anti Tropomyosin of the embryo protein extracts before taken to the IP with Slit, is shown in the lower panel, indicating comparable protein levels in each of the samples.</p