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ERK1 and ERK2 MAPK are key regulators of distinct gene sets in zebrafish embryogenesis.

ABSTRACT: BACKGROUND: The MAPK signaling proteins are involved in many eukaryotic cellular processes and signaling networks. However, specific functions of most of these proteins in vertebrate development remain elusive because of potential redundancies. For instance, the upstream activation pathways for ERK1 and ERK2 are highly similar, and also many of their known downstream targets are common. In contrast, mice and zebrafish studies indicate distinct roles for both ERKs in cellular proliferation, oncogenic transformation and development. A major bottleneck for further studies is that relatively little is known of in vivo downstream signaling specific for these kinases. RESULTS: Microarray based gene expression profiling of ERK1 and ERK2 knockdown zebrafish embryos at various stages of early embryogenesis resulted in specific gene expression signature sets that showed pronounced differences in gene ontology analyses. In order to predict functions of these genes, zebrafish specific in silico signaling pathways involved in early embryogenesis were constructed using the GenMAPP program. The obtained transcriptome signatures were analyzed in the BMP, FGF, Nodal and Wnt pathways. Predicted downstream effects of ERK1 and ERK2 knockdown treatments on key pathways responsible for mesendoderm development were confirmed by whole mount in situ hybridization experiments. CONCLUSION: The gene ontology analyses showed that ERK1 and ERK2 target common and distinct gene sets, confirming the difference in knockdown phenotypes and diverse roles for these kinases during embryogenesis. For ERK1 we identified specific genes involved in dorsal-ventral patterning and subsequent embryonic cell migration. For ERK2 we identified genes involved in cell-migration, mesendoderm differentiation and patterning. The specific function of ERK2 in the initiation, maintenance and patterning of mesoderm and endoderm formation was biologically confirmed.

SUBMITTER: Krens SF

PROVIDER: S-EPMC2390552 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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ERK1 and ERK2 MAPK are key regulators of distinct gene sets in zebrafish embryogenesis.

Krens S F Gabby SF Corredor-Adámez Maximiliano M He Shuning S Snaar-Jagalska B Ewa BE Spaink Herman P HP

BMC genomics 20080428

<h4>Background</h4>The MAPK signaling proteins are involved in many eukaryotic cellular processes and signaling networks. However, specific functions of most of these proteins in vertebrate development remain elusive because of potential redundancies. For instance, the upstream activation pathways for ERK1 and ERK2 are highly similar, and also many of their known downstream targets are common. In contrast, mice and zebrafish studies indicate distinct roles for both ERKs in cellular proliferation ...[more]

PMID: 18442396

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Project description:BackgroundThe Ras/Raf/MEK/ERK signaling pathway is involved in essential cell processes and it is abnormally activated in ~30 % of cancers and cognitive disorders. Two ERK isoforms have been described, ERK1 and ERK2; ERK2 being regarded by many as essential due to the embryonic lethality of ERK2 knock-out mice, whereas mice lacking ERK1 are viable and fertile. The controversial question of why we have two ERKs and whether they have differential functions or display functional redundancy has not yet been resolved.ResultsTo investigate this question we used a novel approach based on comparing the evolution of ERK isoforms' sequences and protein expression across vertebrates. We gathered and cloned erk1 and erk2 coding sequences and we examined protein expression of isoforms in brain extracts in all major clades of vertebrate evolution. For the first time, we measured each isoforms' relative protein level in phylogenetically distant animals using anti-phospho antibodies targeting active ERKs. We demonstrate that squamates (lizards, snakes and geckos), despite having both genes, do not express ERK2 protein whereas other tetrapods either do not express ERK1 protein or have lost the erk1 gene. To demonstrate the unexpected squamates' lack of ERK2 expression, we targeted each ERK isoform in lizard primary fibroblasts by specific siRNA-mediated knockdown. We also found that undetectable expression of ERK2 in lizard is compensated by a greater strength of lizard's erk1 promoter. Finally, phylogenetic analysis revealed that ERK1 amino acids sequences evolve faster than ERK2's likely due to genomic factors, including a large difference in gene size, rather than from functional differences since amino acids essential for function are kept invariant.ConclusionsERK isoforms appeared by a single gene duplication at the onset of vertebrate evolution at least 400 Mya. Our results demonstrate that tetrapods can live by expressing either one or both ERK isoforms, supporting the notion that ERK1/2 act interchangeably. Substrate recognition sites and catalytic cleft are nearly invariant in all vertebrate ERKs further suggesting functional redundancy. We suggest that future ERK research should shift towards understanding the role and regulation of total ERK quantity, especially in light of newly described erk2 gene amplification identified in tumors.

Project description:The zebrafish (Danio rerio) is an established model organism for developmental and biomedical research. It is frequently used for high-throughput functional genomics experiments, such as genome-wide gene expression measurements, to systematically analyze molecular mechanisms. However, the use of whole embryos or larvae in such experiments leads to a loss of the spatial information. To address this problem, we have developed a tool called Zebrafish Expression Ontology of Gene Sets (ZEOGS) to assess the enrichment of anatomical terms in large gene sets. ZEOGS uses gene expression pattern data from several sources: first, in situ hybridization experiments from the Zebrafish Model Organism Database (ZFIN); second, it uses the Zebrafish Anatomical Ontology, a controlled vocabulary that describes connected anatomical structures; and third, the available connections between expression patterns and anatomical terms contained in ZFIN. Upon input of a gene set, ZEOGS determines which anatomical structures are overrepresented in the input gene set. ZEOGS allows one for the first time to look at groups of genes and to describe them in terms of shared anatomical structures. To establish ZEOGS, we first tested it on random gene selections and on two public microarray datasets with known tissue-specific gene expression changes. These tests showed that ZEOGS could reliably identify the tissues affected, whereas only very few enriched terms to none were found in the random gene sets. Next we applied ZEOGS to microarray datasets of 24 and 72 h postfertilization zebrafish embryos treated with beclomethasone, a potent glucocorticoid. This analysis resulted in the identification of several anatomical terms related to glucocorticoid-responsive tissues, some of which were stage-specific. Our studies highlight the ability of ZEOGS to extract spatial information from datasets derived from whole embryos, indicating that ZEOGS could be a useful tool to automatically analyze gene expression pattern features of any large zebrafish gene set.

Project description:Lymphoid neoplasms form a family of cancers affecting B-cells, T-cells, and NK cells. The Toll-Like Receptor (TLR) signaling adapter molecule MYD88 is the most frequently mutated gene in these neoplasms. This signaling adaptor relays signals from TLRs to downstream effector pathways such as the Nuclear Factor kappa B (NF?B) and Mitogen Activated Protein Kinase (MAPK) pathways to regulate innate immune responses. Gain-of-function mutations such as MYD88[L265P] activate downstream signaling pathways in absence of cognate ligands for TLRs, resulting in increased cellular proliferation and survival. This article reports an analysis of non-synonymous somatic mutations found in the TLR signaling network in lymphoid neoplasms. In accordance with previous reports, mutations map to MYD88 pro-inflammatory signaling and not TRIF-mediated Type I IFN production. Interestingly, the analysis of somatic mutations found downstream of the core TLR-signaling network uncovered a strong association with the ERK1/2 MAPK cascade. In support of this analysis, heterologous expression of MYD88[L265P] in HEK293 cells led to ERK1/2 MAPK phosphorylation in addition to NF?B activation. Moreover, this activation is dependent on the protein kinase Tumor Promoting Locus 2 (TPL2), activated downstream of the IKK complex. Activation of ERK1/2 would then lead to activation, amongst others, of MYC and hnRNPA1, two proteins previously shown to contribute to tumor formation in lymphoid neoplasms. Taken together, this analysis suggests that TLR-mediated ERK1/2 activation via TPL2 may be a novel path to tumorigenesis. Therefore, the hypothesis proposed is that inhibition of ERK1/2 MAPK activation would prevent tumor growth downstream of MYD88[L265]. It will be interesting to test whether pharmacological inhibitors of this pathway show efficacy in primary tumor cells derived from hematologic malignancies such as Waldenstrom's Macroglobulinemia, where the majority of the cells carry the MYD88[L265P] mutation.

Dataset Information

ERK1 and ERK2 MAPK are key regulators of distinct gene sets in zebrafish embryogenesis.

Publications

ERK1 and ERK2 MAPK are key regulators of distinct gene sets in zebrafish embryogenesis.

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