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Shoc2 Mediates Hematopoietic Signals of the ERK1/2 Pathway
Author(s) -
JANG HYEIN,
Jang Eun Ryoung,
Morris Ann,
ForbesOsborne Marrie,
Galperin Emilia
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.929.4
Subject(s) - biology , zebrafish , microbiology and biotechnology , gene , genetics , cancer research
Activity of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) pathway plays a critical role in normal human development. Aberrant ERK1/2 signaling caused by germline mutations in genes of the Ras‐mediated ERK1/2 pathway results in developmental disorders cumulatively called ‘RASopathies’. Shoc2, the scaffold protein in the ERK1/2 pathway, accelerates ERK1/2 activity by bringing the signaling components of the pathway, Ras and Raf‐1, to close proximity. Noonan‐like RASopathy causing Shoc2 mutations emphasize the significance of ERK1/2 signaling transmitted through the Shoc2 scaffolding module. However, the physiological role of Shoc2 and the specificity of the signals transduced through the Shoc2 scaffolding complex remain largely unknown. In our work, we took advantage of the vertebrate zebrafish model that is ideally suited for developmental genetic studies due to its optical transparency and ex vivo development. To delineate the developmental ERK1/2 signals mediated through the Shoc2 complex, a translation‐blocking shoc2 morpholino (MO) was injected into 1–2 cell stage zebrafish embryos. The outcomes of the Shoc2 depletion were examined using biochemical methods such as Western blot analysis, RT‐PCR and whole embryo staining/imaging. We found that Shoc2 depletion in zebrafish embryos results in decreased ERK1/2 activity. Shoc2 MO‐injected embryos also possessed a larger yolk, kinked tail, and an enlarged hindbrain. Importantly, Shoc2 morphants displayed dramatically reduced numbers of circulating blood cells and loss of erythropoietic and myeloid lineages. To further understand the role of Shoc2 in differentiation of hematopoietic stem cells, we utilized CRISPR/Cas9 gene editing to generate the Shoc2 zebrafish knockout. 12 germ‐line transmitted alleles were recovered from the mutagenesis. All alleles had lesions in similar locations, causing frame‐shifts around amino acid 225 and introducing aberrant stop codons (TAA or TAG) prior to termination. Our data demonstrate that Shoc2 depletion affects both waves of hematopoiesis: primitive and definitive. Shoc2 CRISPR zebrafish knockout will now be utilized to examine quantitative changes in the hematopoiesis‐specific ERK1/2 transcriptional program. Support or Funding Information Funding sources: National Institute of General Medical Siences, American Cancer Society, American Heart Association