Maternal inheritance of twist and analysis of MAPK activation in embryos of the polychaete Annelid Platynereis dumerilii

In this study, we aimed to identify molecular mechanisms involved in the specification of the 4d (mesentoblast) lineage in Platynereis dumerilii. We employ RT-PCR and in situ hybridization against the Platynereis dumerilii twist homolog (Pdu-twist) to reveal mesodermal specification within this lineage. We show that Pdu-twist mRNA is already maternally distributed. After fertilization, ooplasmatic segregation leads to relocation of Pdu-twist transcripts into the somatoblast (2d) lineage and 4d, indicating that the maternal component of Pdu-twist might be an important prerequisite for further mesoderm specification but does not represent a defining characteristic of the mesentoblast. However, after the primordial germ cells have separated from the 4d lineage, zygotic transcription of Pdu-twist is exclusively observed in the myogenic progenitors, suggesting that mesodermal specification occurs after the 4d stage. Previous studies on spiral cleaving embryos revealed a spatio-temporal correlation between the 4d lineage and the activity of an embryonic organizer that is capable to induce the developmental fates of certain micromeres. This has raised the question if specification of the 4d lineage could be connected to the organizer activity. Therefore, we aimed to reveal the existence of such a proposed conserved organizer in Platynereis employing antibody staining against dpERK. In contrast to former observations in other spiralian embryos, activation of MAPK signaling during 2d and 4d formation cannot be detected which questions the existence of a conserved connection between organizer function and specification of the 4d lineage. However, our experiments unveil robust MAPK activation in the prospective nephroblasts as well as in the macromeres and some micromeres at the blastopore in gastrulating embryos. Inhibition of MAPK activation leads to larvae with a shortened body axis, defects in trunk muscle spreading and improper nervous system condensation, indicating a critical function for MAPK signaling for the reorganization of embryonic tissues during the gastrulation process

Entwicklungsgenetische Analyse der Differenzierungsprozesse im viszeralen Mesoderm von Drosophila melanogaster

Der larvale Mitteldarm der Taufliege Drosophila melanogaster wird während der Embryonalentwicklung gebildet und besteht aus dem entodermalen Darmepithel, der viszeralen Muskulatur sowie der dazwischen liegenden extrazellulären Matrix (ECM). Bisherige Studien zur Mitteldarmentwicklung konzentrierten sich vor allem auf die Rolle der viszeralen Muskulatur, mögliche Einflüsse des Entoderms und der ECM wurden dagegen kaum untersucht. Mit Hilfe des Allels 3B1-038, das aufgrund eines charakteristischen viszeralen Phänotyps identifiziert wurde (Stute et al., 2004), konnten diese Aspekte nun erstmals analysiert werden. Die Komplementationsanalyse von 3B1-038 offenbart zwei unabhängige Mutationen auf dem betroffenen Chromosom, die sich beide auf die viszerale Muskeldifferenzierung auswirken. In Anlehnung an die charakteristischen viszeralen Phänotypen wurden die entsprechenden Allele „gürtelchen“ (gurt) und „knödel“ (knod) genannt. Die morphologische Analyse von gurt enthüllt weder Defekte während der Frühentwicklung des viszeralen Mesoderms noch während der Myoblastenfusion. Allerdings unterbleibt die Streckung der viszeralen Myotuben während der späteren Myogenese. Weitere Analysen decken den Verlust der Mitteldarmzellen in mutanten Embryonen auf, was schließlich zur Identifikation von gurt als Allel der entodermalen Determinante huckebein (hkb) führt. Interessanterweise werden charakteristische Streckungsdefekte in der viszeralen Muskulatur auch durch Mutationen in anderen entodermalen Selektorgenen hervorgerufen, was einen bislang unbekannten Einfluss des Entoderms auf die viszerale Muskeldifferenzierung aufdeckt. Durch die Identifizierung des viszeralen Allels knödel als neues Null-Allel des LamininB2 (LanB2)-Gens, konnte darüber hinaus der Einfluss der ECM auf die Mitteldarmentwicklung untersucht werden. Der Verlust von LanB2 beeinträchtigt die extrazelluläre Verteilung von anderen ECM-Komponenten und verhindert die Bildung einer Basallamina (BM). In mutanten Embryonen kommt es außerdem zur Fehlentwicklung des Mitteldarms, des Herzens, des Nervensystems, der Tracheen, der Körpermuskulatur und des Fettkörpers. Im Zuge der Mitteldarmentwicklung beeinflusst Laminin die gerichtete Migration von Entoderm und longitudinalen Muskelgründerzellen. In späteren Stadien kommt es zum Verlust der epithelialen Polarität in den Entodermzellen, zu Streckungsdefekten in den viszeralen Myotuben und zur abnormalen Entwicklung der Magenblindsäcke und der Cardia. Das Drosophila-Genom kodiert für vier Lamininuntereinheiten, die sich zu zwei unterschiedlichen Heterotrimeren zusammenlagern. Die hier durchgeführten Experimente legen nahe, dass beide Trimere während der Mitteldarmentwicklung wirken und unterschiedliche Rollen während des Zusammenbaus der Basallamina und der Gewebeadhäsion übernehmen. Weitere Analysen decken eine genetische Interaktion zwischen LanB2 und dem NG2/CSPG4-verwandten kon-tiki (kon) sowie Thrombospondin TSP) in der somatischen Muskulatur auf, was auf eine Funktion von Laminin während der Muskelanheftung und dem Erhalt der Muskulatur hindeutet.The larval midgut of the fruit fly Drosophila melanogaster is established during embryogenesis and consists of an inner endodermal epithelium and the visceral musculature, both embedded in the extracellular matrix (ECM). Previous studies provided detailed insights into the “mesodermal aspects” of midgut development while possible influences from the endoderm and the ECM have been barely investigated. To address this point I analysed the allele 3B1-038 which had been identified due to a characteristic visceral phenotype (Stute et al., 2004). Deficiency mapping uncovers two independent mutations on the affected chromosome, that both impair visceral muscle differentiation. According to their distinct visceral phenotypes, the corresponding alleles were named “gürtelchen” (gurt) and “knödel” (knod). Further analysis of gurt mutant embryos reveals that early visceral mesoderm development and myoblast fusion appear unaffected. However, at the end of embryogenesis visceral myotube elongation is completely blocked and two belt-shaped muscle strands flank the central yolk. Examination of the endoderm development uncovers the complete loss of midgut cells in mutant embryos. gurt is finally identified as novel allele of the terminal gap gene huckebein (hkb) which is pivotal for endoderm determination. Interestingly, visceral muscle differentiation is also impaired in the absence of other endodermal selector genes. These results enlighten a previously unconsidered aspect of visceral muscle development. Investigating a role of the ECM during midgut development I took advantage of the knödel (knod)allele that turned out to be a novel null mutation in the LamininB2 (LanB2) gene. The loss of LamininB2 affects extracellular deposition of other ECM-components and induces the breakdown of basement membranes (BM). As further consequence, LanB2 mutant embryos exhibit phenotypes in a wide range of tissues including midgut, heart, trachea, nervous system, fat body and somatic musculature. The visceral phenotype is characterized by defects during endoderm migration, midgut epithelium formation, guided migration and stretching of visceral myotubes as well as an abnormal morphology of gastric caeca and cardia. The Drosophila genome encodes for four laminin subunits that self-assemble into two distinct heterotrimers. Comparative analyses reveal a redundant function of both trimers during midgut development but also their individual roles during BM self-assembly and tissue adhesion. Finally, LanB2 interacts genetically with the NG2/CSPG4-homolog kon-tiki and Thrombospondin in the somatic musculature, indicating an influence of laminin on muscle attachment and maintenance of muscle tendon sites

Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm

In this study we describe the morphological and genetic analysis of the Drosophila mutant gurtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein(gurtelchen) (hkb(gurt)) mutant embryos. It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts; play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. our analyses of the visceral phenotype of hkb(gurt) mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion. (C) 2009 Elsevier Ireland Ltd. All rights reserve

Inhibition of MAPK activity causes defects in muscle and nervous system development.

<p>Embryos were treated with 10 µM, 25 µM or 50 µM of the MEK inhibitor U0126. Control groups were incubated in 0.5% DMSO/NSW or pasteurized NSW from 13.5 to 16.5 hpf. All treated embryos from a clutch were collected and fixed 66 hpf for further analysis. <b>A–A″′, C–C″′, E–E″′.</b> Musculature of larvae was labeled with FITC-Phalloidin (actin in green or white), the nervous system was stained with an antibody against acetylated tubulin (aat in red or white), Hoechst labeling of DNA appears blue. <b>B, D</b> and <b>F</b> represent <i>Pdu-Mhc</i> detections (blue) after <i>in situ</i> hybridization. <b>A–A″′.</b> Normally developed muscles and nerves in a 66 hpf larvae of the 0.5% DMSO control group were classified as phenotype 0 (P0). <b>B.... </b><i>Pdu-Mhc</i> expression in a P0 larva at 66 hpf. <b>C–C’’’.</b> Larvae with a shortened body axis and reduced parapodial development were classified as phenotype 1 (P1). Muscle pattern defects occur due to abnormal positioned and orientated muscles. The nervous system is formed, but nerve fibers are messily arranged. <b>D.... </b><i>Pdu-Mhc</i> expression in a P1 larva. <b>E–E’’’.</b> Larvae classified as phenotype 2 (P2) lack a secondary body axis, fail to elongate and appear rounded in shape. Muscle accumulation is clearly observed and fibers of the ventral nervous system are missing. <b>F.... </b><i>Pdu-Mhc</i> expression in a P2 larva. <b>G.</b> Quantification of the proportions of non-developed eggs (n.d.), affected (phenotypes 1&2) and unaffected (phenotype 0) larvae in treatment and control groups. Means and standard errors of means are shown. Significance levels revealed by the Tukey HSD post hoc test are indicated for selected groups (*  = p<0.05, **  = p<0.01, n.s.  =  not significant/p>0.05). Data were obtained from three experimental replicates. Total Numbers (n) of counted larvae: NSW: n = 913, 0.5% DMSO: n = 1545, 10 µM: n = 1387, 25 µM: n = 1351 and 50 µM: n = 1503. <b>H–I.... </b><i>Pdu-twist</i> expression in control (H) and U0126 treated larva (I) at 24 hpf. Improper positioning of <i>Pdu-twist</i> expressing cells was observed in 45 of 310 larvae after 0.5% DMSO vehicle treatment and 161 of 270 larvae after treatment with 10 µM U0126. Scale bars are 50 µm and 10 µm for whole embryos and close ups, respectively.</p

MAPK activation during early development in <i>Platynereis dumerilii</i>.

<p>Antibody staining against di-phosphorylated, activated MAPK/ERK (dpERK) in red or white, DNA-staining with Hoechst appears in blue, actin was marked by FITC-coupled Phalloidin (in green). <b>A, B</b> Embryos at the 38- and the 46-cell stage exhibit no dpERK staining in the mesentoblast (4d) and its descendants ML and MR. <b>C.</b> Initial dpERK staining was detected within the nephroblasts (n) in the animal hemisphere of a 7.5 hpf early blastula. <b>D.</b> MAPK activation is still visible during further head kidney development in the mid-blastula (10.5 hpf). <b>E–G″.</b> dpERK staining is visible during gastrulation in nuclei of small cells (arrowheads) and macromeres (M) in the region of the blastopore. <b>F–F′.</b> Micromeres with MAPK activity show an accumulation of filamentous actin at 15 hpf. <b>G′–G″.</b> MAPK positive cells in the region of the blastopore at 15 hpf. <b>G″′.</b> dpERK positive macromere nuclei in the same embryo as in G but different focal plane. <b>H, H′. </b><i>Pdu-twist in situ</i> hybridization in combination with dpERK staining in a 15 hpf embryo. Activated MAPK and <i>Pdu-twist</i> positive cells are in close proximity at the region of the blastopore (asterisk). Arrows point towards two dpERK-positive nuclei that are in the same focal plane as the nuclei (arrowheads) of two <i>Pdu-twist</i> (black) expressing cells. Scale bars are 50 µm and 10 µm in whole embryo views and close-ups, respectively.</p

Identification of the Wallenda JNKKK as an Alk suppressor reveals increased competitiveness of Alk-expressing cells

Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase of the insulin receptor super-family that functions as oncogenic driver in a range of human cancers such as neuroblastoma. In order to investigate mechanisms underlying Alk oncogenic signaling, we conducted a genetic suppressor screen in Drosophila melanogaster. Our screen identified multiple loci important for Alk signaling, including members of Ras/Raf/ERK-, Pi3K-, and STAT-pathways as well as tailless (tll) and foxo whose orthologues NR2E1/TLX and FOXO3 are transcription factors implicated in human neuroblastoma. Many of the identified suppressors were also able to modulate signaling output from activated oncogenic variants of human ALK, suggesting that our screen identified targets likely relevant in a wide range of contexts. Interestingly, two misexpression alleles of wallenda (wnd, encoding a leucine zipper bearing kinase similar to human DLK and LZK) were among the strongest suppressors. We show that Alk expression leads to a growth advantage and induces cell death in surrounding cells. Our results suggest that Alk activity conveys a competitive advantage to cells, which can be reversed by over-expression of the JNK kinase kinase Wnd

The Zic family homologue Odd-paired regulates Alk expression in Drosophila

The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression

FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase

Aberrant activation of anaplastic lymphoma kinase (ALK) has been described in a range of human cancers, including non-small cell lung cancer and neuroblastoma (Hallberg and Palmer, 2013). Vertebrate ALK has been considered to be an orphan receptor and the identity of the ALK ligand(s) is a critical issue. Here we show that FAM150A and FAM150B are potent ligands for human ALK that bind to the extracellular domain of ALK and in addition to activation of wild-type ALK are able to drive 'superactivation' of activated ALK mutants from neuroblastoma. In conclusion, our data show that ALK is robustly activated by the FAM150A/B ligands and provide an opportunity to develop ALK-targeted therapies in situations where ALK is overexpressed/activated or mutated in the context of the full length receptor