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

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

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

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

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

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm

Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis