Muscle formation during embryogenesis of the polychaete (Dorvilleidae) – new insights into annelid muscle patterns-0

<p><b>Copyright information:</b></p><p>Taken from "Muscle formation during embryogenesis of the polychaete (Dorvilleidae) – new insights into annelid muscle patterns"</p><p>http://www.frontiersinzoology.com/content/5/1/1</p><p>Frontiers in Zoology 2008;5():1-1.</p><p>Published online 2 Jan 2008</p><p>PMCID:PMC2254616.</p><p></p>rostomium are the ventral diagonal muscles (dVLM). Two transverse muscles are present in the prostomium: PStm2 crosses the dVLM dorsally, whereas PStm1 lies between the dorsal and the ventral longitudinal muscles. The complex musculature of the ventrally situated pharynx (phar) is heavily stained. The inner strands of the dorsal longitudinal muscles (DLM) cross repeatedly at the dorsal midline (circles). Four pairs of parapodia are present, with prominent parapodial muscle complexes (pmc). Transverse (tm) and diagonal muscles (dm) cross the dorsal side irregularly. Several transverse muscles surround the posterior zone of segment proliferation (arrowhead). The last transverse muscle circle marks the pygidium (tagged arrow). . Ventral view, phalloidin staining (red) and anti-acetylated-tubulin staining (green). The ciliary bands are stained by anti-acetylated tubulin antibody. These are the prototroch (prot) of the prostomium, the metatroch (met) and the ciliary band of the segment like annulus within the peristomium (pcb), and the four ciliary bands of the chaetigers (1–4 BS) anterior to the telotroch (tel) of the pygidium. The boxed region marks the close-up depicted in Figure C (vnc-ventral nerve cord, VLM-ventral longitudinal muscle) . Close-up from B. Ventral view, phalloidin staining and anti-acetylated tubulin antibody depth coded (in μm). Upper panel shows the ventral nerve cord (vnc), lower panel shows the median ventral longitudinal muscle (mVLM). Depth coding indicates that the mVLM is situated dorsal (deep) to the vnc (circles). . Histological cross section through chaetiger of an adult. The dorsal longitudinal muscles (DLM) lie just below the epidermis (epi). The uniramous parapodia (ppd) are ventrolaterally situated. The ventral longitudinal muscles (VLM) lie on either side of the ventral nerve cord (vnc). . Close up from D. Same labeling as in D, additionally labeled are the dorsally situated transverse muscles (tm). . Close up from E depicting the dorsal longitudinal muscle (DLM) and a transverse muscle (tm) lying above it (supralongitudinally). . 3D-reconstruction from a series of histological sections in the same body-region as shown in D. Anterior is toward the top of the figure. Depicted are the dorsal longitudinal muscles (DLM) with their dorsomedian crossing strands (arrow), diagonal muscles (dm) and transverse muscles (tm) in a supralongitudinal position. The ventral longitudinal muscles (VLM) and the median ventral longitudinal muscle (mVLM) surround the ventral nerve cord (vnc – only the main tracts of the neuropile shown here). The anterior and posterior dorsal parapodial muscles (adPM, pdPM) stretch diagonally toward posterior and anterior respectively, underneath the dorsal longitudinal muscles

Muscle formation during embryogenesis of the polychaete (Dorvilleidae) – new insights into annelid muscle patterns-1

<p><b>Copyright information:</b></p><p>Taken from "Muscle formation during embryogenesis of the polychaete (Dorvilleidae) – new insights into annelid muscle patterns"</p><p>http://www.frontiersinzoology.com/content/5/1/1</p><p>Frontiers in Zoology 2008;5():1-1.</p><p>Published online 2 Jan 2008</p><p>PMCID:PMC2254616.</p><p></p>rostomium are the ventral diagonal muscles (dVLM). Two transverse muscles are present in the prostomium: PStm2 crosses the dVLM dorsally, whereas PStm1 lies between the dorsal and the ventral longitudinal muscles. The complex musculature of the ventrally situated pharynx (phar) is heavily stained. The inner strands of the dorsal longitudinal muscles (DLM) cross repeatedly at the dorsal midline (circles). Four pairs of parapodia are present, with prominent parapodial muscle complexes (pmc). Transverse (tm) and diagonal muscles (dm) cross the dorsal side irregularly. Several transverse muscles surround the posterior zone of segment proliferation (arrowhead). The last transverse muscle circle marks the pygidium (tagged arrow). . Ventral view, phalloidin staining (red) and anti-acetylated-tubulin staining (green). The ciliary bands are stained by anti-acetylated tubulin antibody. These are the prototroch (prot) of the prostomium, the metatroch (met) and the ciliary band of the segment like annulus within the peristomium (pcb), and the four ciliary bands of the chaetigers (1–4 BS) anterior to the telotroch (tel) of the pygidium. The boxed region marks the close-up depicted in Figure C (vnc-ventral nerve cord, VLM-ventral longitudinal muscle) . Close-up from B. Ventral view, phalloidin staining and anti-acetylated tubulin antibody depth coded (in μm). Upper panel shows the ventral nerve cord (vnc), lower panel shows the median ventral longitudinal muscle (mVLM). Depth coding indicates that the mVLM is situated dorsal (deep) to the vnc (circles). . Histological cross section through chaetiger of an adult. The dorsal longitudinal muscles (DLM) lie just below the epidermis (epi). The uniramous parapodia (ppd) are ventrolaterally situated. The ventral longitudinal muscles (VLM) lie on either side of the ventral nerve cord (vnc). . Close up from D. Same labeling as in D, additionally labeled are the dorsally situated transverse muscles (tm). . Close up from E depicting the dorsal longitudinal muscle (DLM) and a transverse muscle (tm) lying above it (supralongitudinally). . 3D-reconstruction from a series of histological sections in the same body-region as shown in D. Anterior is toward the top of the figure. Depicted are the dorsal longitudinal muscles (DLM) with their dorsomedian crossing strands (arrow), diagonal muscles (dm) and transverse muscles (tm) in a supralongitudinal position. The ventral longitudinal muscles (VLM) and the median ventral longitudinal muscle (mVLM) surround the ventral nerve cord (vnc – only the main tracts of the neuropile shown here). The anterior and posterior dorsal parapodial muscles (adPM, pdPM) stretch diagonally toward posterior and anterior respectively, underneath the dorsal longitudinal muscles

Embryonic expression and localization of Loh and Prc.

<p>(<b>A–F</b>) Whole mount <i>in situ</i> hybridization revealing the expression patterns of <i>loh</i> (A–C) and <i>prc</i> (D–F) during embryogenesis. Chordotonal organs (double arrowhead in C), the ring gland (arrowhead in E, F) and oenocytes (double arrowhead in F) are indicated. (<b>G, H</b>) Double labeling of <i>loh</i> transcripts and Tinman (G) or <i>odd</i>-LacZ (H) in stage 17 embryos. (<b>I, J</b>) Co-staining of either Prc (I) or Loh (J) with Vkg::GFP in stage 17 embryos demonstrates localization within the cardiac ECM at the basal side of cardiomyocytes. (<b>K, L</b>) Co-staining of Loh and Prc shows co-localization of both proteins.</p

Loss of pericardial cell adhesion causes loss of heart function.

<p>(<b>A</b>) Scheme depicting the basics of dye angiography in pharate adults. The main body parts, the dorsal vessel (DV) and the injection area are indicated. (<b>B</b>) Head of a wild type animal (corresponding to dashed box in scheme A) showing the accumulation of the tracer at four consecutive time points after injection. (<b>C</b>) Mean pixel intensities measured at four consecutive time points showing cardiac output in wild type (<i>white¹¹¹⁸</i>) and homozygous <i>prc^MB03017</i> and <i>loh^MB05750</i> pharate adult animals. Error bars are s.e.m. The region used for measurement is indicated in the lowest panel in B.</p

Isolation of new heart integrity mutants.

<p>(<b>A–D</b>) Combinations of <i>loh</i> mutant alleles, cardiac cells are marked with <i>hand</i>C-GFP. All transheterozygous mutant larvae display the detachment of pericardial cells (arrowheads) from the heart tube. (<b>E, F</b>) Pericardial cell detachment in transheterozygous <i>prc</i> mutant larvae (arrowhead in F). Pericardial cells were stained using toluidine blue (TB).</p

The Conserved ADAMTS-like Protein Lonely heart Mediates Matrix Formation and Cardiac Tissue Integrity

<div><p>Here we report on the identification and functional characterization of the ADAMTS-like homolog <i>lonely heart</i> (<i>loh</i>) in <i>Drosophila melanogaster</i>. Loh displays all hallmarks of ADAMTSL proteins including several thrombospondin type 1 repeats (TSR1), and acts in concert with the collagen Pericardin (Prc). Loss of either <i>loh</i> or <i>prc</i> causes progressive cardiac damage peaking in the abolishment of heart function. We show that both proteins are integral components of the cardiac ECM mediating cellular adhesion between the cardiac tube and the pericardial cells. Loss of ECM integrity leads to an altered myo-fibrillar organization in cardiac cells massively influencing heart beat pattern. We show evidence that Loh acts as a secreted receptor for Prc and works as a crucial determinant to allow the formation of a cell and tissue specific ECM, while it does not influence the accumulation of other matrix proteins like Nidogen or Perlecan. Our findings demonstrate that the function of ADAMTS-like proteins is conserved throughout evolution and reveal a previously unknown interaction of these proteins with collagens.</p></div

Prc becomes secreted by the larval fat body and recruited to the heart.

<p>(<b>A, B</b>) Embryonic (stage 17) and larval (L1) expression pattern of the <i>prc</i>>GFP reporter. (<b>C, D</b>) Prc protein becomes trapped in adipocytes of <i>prc</i>><i>sar1</i>-IR knock down first instar larvae, proving that the protein becomes expressed by the fat body. Scale bar is 10 µm. (<b>E</b>) Immunoblots of whole larval extracts probed against Prc. While <i>hand</i>C-Gal4 driven knock down does not alter the total amount of Prc the protein is nearly undetectable in <i>prc</i>-Gal4 driven knock down animals. Total protein was stained with amido black 10B. (<b>F</b>) Percentage of third instar larvae showing pericardial cell detachment induced by <i>prc</i> knock down either driven by <i>hand</i>C-Gal4 or <i>prc</i>-Gal4. <i>n</i> indicates the total number of tested animals. (<b>G</b>) Scheme of Prc matrix formation. Secreted Prc from different sources (heart cells in the embryo, adipocytes in the larva) becomes incorporated into the cardiac ECM dependent on Loh.</p

Localization of Prc depends on Loh but not vice versa.

<p>(<b>A–I</b>) Localization of Perlecan and Prc in stage 17 control embryos (A–C), compared to homozygous <i>loh^MB05750</i> (D–F) or homozygous <i>prc^MB03017</i> mutants (G–I). Prc but not Perlecan becomes mis-localized by the absence of Loh. (<b>J–O</b>) Localization of Loh in stage 17 control embryos (J–L) compared to homozygous <i>prc^MB03017</i> mutants (M–O). The localization of Loh to the ECM is not affected by the absence of Prc. Of note, the anti-Loh antiserum needs heat fixation leading to a different appearance of Prc in the stained control animals compared to chemical fixation as shown in A. (<b>P–R</b>) The luminal ECM of cardiomyocytes at embryonic stage 17 is not altered in either homozygous <i>loh^MB05750</i> or <i>prc^MB03017</i> mutants. The arrowheads indicate the thickness of the ECM. (<b>S</b>) Quantification of luminal basement membrane (BM) thickness in animals of the indicated genotypes. Mutants do not show significant alterations in ECM thickness. Error bars shown are standard deviation (s.d.) (<b>T–V</b>) TEM section of the adhesion area between cardiomyocytes (CC) and pericardial cells (PC). Lack of either <i>loh</i> or <i>prc</i> cause gaps between the cells. Scale bars are 250 nm.</p

Molecular characterization of <i>loh</i> and <i>prc</i>.

<p>(<b>A</b>) Developmental Northern blots showing <i>loh</i> and <i>prc</i> expression in total RNA samples of 0–24 h old embryos (E), first, second or third instar larvae (L1–L3), mid-stage pupae (P) or adults (A) using gene specific riboprobes (indicated in B and C). (<b>B, C</b>) Schematic representation of <i>loh</i> (B) and <i>prc</i> (C) gene loci and transcripts. The schemes indicate the position of transposons, location of hairpins (IR) used for knock down and riboprobes used for Northern analysis (NB) and <i>in situ</i> hybridization (ISH). (<b>D</b>) Immunoblot of total protein extracts obtained from stage 17 control, homozygous Df(2L)Exel7048 or homozygous <i>loh^MB05750</i> embryos probed with antibodies against Loh or βTub. Loh is undetectable in homozygous deficiency or mutant extracts. (<b>E</b>) Immunoblot of total protein extracts obtained from control or homozygous <i>prc^MB03017</i> 0–24 h old embryos (E), third instar larvae (L3) mid-stage pupae (P) or adults (A) probed with antibodies against Prc or βTub. Prc is undetectable in extracts of homozygous mutants.</p

Loh induces Prc matrix formation <i>in vivo</i>.

<p>(<b>A</b>) Prc (green) is absent from the ECM of larval muscles or the fat body in wild type third instar larvae. (<b>B</b>) Schematic representation of ectopically expressed LohA wild type protein or the secretion defective variant LohA^ΔSP. (<b>C</b>) Expression of LohA (red) in adipocytes or myocytes results in the formation of a dense Prc matrix (green) along both cell types in third instar larvae, while secretion of LohA is essential to mediate Prc recruitment. Inset shows accumulation of LohA at muscle attachment sites. (<b>D</b>) Loh and Prc partially co-localize in an artificial matrix around adipocytes. Loh distributes as weak fibers accumulating in a spotted fashion along cellular contacts (arrowheads). (<b>E</b>) Single optical slice of adipocytes. Loh co-localizes to Prc (arrowhead) at the root of Prc fibers (double arrowhead). (<b>F</b>) Loh co-immunoprecipitates with Prc and vice versa from total adult protein extracts demonstrating a biochemical interaction of both proteins.</p