During vertebrate gastrulation both concurrent inductive events and cell movements are

During vertebrate gastrulation both concurrent inductive events and cell movements are required for axis formation. or knockdown can rescue convergent extension defects induced by overexpression or knockdown respectively. Therefore is an essential novel regulator for normal convergent and extension movements by regulating mitogen-activated protein kinase (MAPK) JNK signaling. INTRODUCTION During vertebrate gastrulation the embryonic body Ergonovine maleate plan is established by coordinated movements of large populations of cells to generate the ectodermal mesodermal and endodermal layers (22 49 66 A key driving force of gastrulation is convergence and extension (CE) movements. The Ergonovine maleate convergence of cells narrows the germ layers and the embryonic body mediolaterally while extension movement elongates the embryonic tissues from head to tail (23 46 In vertebrates the dorsal axial and paraxial mesoderms the notochordal and somitic mesoderms converge and extend (24). CE movements also occur in the Ergonovine maleate neuroectoderm to narrow and elongate the neural floor plate which then folds appositionally and the neural tube is formed (10). Wnt/PCP and Bmp pathways play important roles in cell movements during gastrulation (19 29 45 57 63 c-Jun N-terminal protein kinase (JNK) the signal transducer and activator of transcription 3 (Stat3) and Prickle1 have also been shown to be required for normal Ergonovine maleate CE movements (5 47 52 70 The regulation of gene expression by microRNAs (miRNAs) plays a critical role in regulating fundamental cellular Ergonovine maleate functions and developmental processes (9 32 37 44 71 Inactivation of miRNA biogenesis by the loss of in zebrafish maternal zygotic (is one of the skeletal muscle-specific miRNAs (8 16 43 67 71 and it regulates the proliferation and differentiation of muscle progenitor cells (7 16 27 is also required for efficient regeneration of neuromuscular synapse after acute nerve injury and deficiency of in the amyotrophic lateral sclerosis (ALS) mouse model accelerates disease progression (68). However the function of in early development of vertebrate embryos has not been reported. In zebrafish mature is processed Slit3 from two pre-miRNA transcripts and is maternally expressed and its transcripts exist throughout the early development of zebrafish embryos. We demonstrate that is essential for normal gastrulation cell movements by regulating JNK2 phosphorylation through inhibition of expression. MATERIALS AND METHODS Zebrafish strains and antibodies. Wild-type embryos were obtained from natural matings of the zebrafish Tuebingen strain. Embryos were maintained in Holtfreter’s solution at 28.5°C and staged morphologically as described previously (28). The expression of enhanced green fluorescent protein (EGFP) Pk1a and phosphorylated and total JNK2 were detected by Western blot analysis using the following antibodies: anti-GFP antibody (M20004L; Abmart) anti-Pk1a antibody (55637; ANA SPEC) anti-p-JNK2 antibody (9251; Cell Signaling Technology) and anti-JNK2 antibody (sc-571; Santa Cruz). Constructs. Total RNAs were extracted from 75%-epiboly stage wild-type embryos using TRIzol reagent (Invitrogen) and reversely transcribed with the ReverTra kit (TOYOBO). The expanded 3′ untranslated region (3′UTR) of zebrafish (“type”:”entrez-nucleotide” attrs :”text”:”NM_183342.2″ term_id :”40254656″ term_text :”NM_183342.2″NM_183342.2) with a 50-nucleotide sequence upstream of the stop codon was amplified by reverse transcription (RT)-PCR using the forward primer 5′-ACAGAAGAGAGGACGGAAAGG-3′ and the reverse primer 5′-AATTCCCTCTCAAAGTGGGC-3′ and then inserted downstream of the open reading frame of EGFP to generate the reporter site on mRNA and binding site within the coding region in frame to the open reading frame. The primers were as follows: for binding sites are underlined and the mutations are italic). The full coding sequence of was amplified using the forward primer 5′-ATGGAGCTGGAGAATCACGG-3′ and the reverse primer 5′-TTATGAAATAATACAGTTTTTGCCTTTC-3′ and then cloned into the pCS-Flag vector. All the sequences were confirmed by DNA sequencing and the expression of was verified by Western blotting using anti-Flag antibody (M20008L; Abmart). Morpholinos microinjection and hybridization. The morpholino (206-MO1) (5′-ACCACACACTTCCTTACATTCCATAACTTG-3′) and morpholino (206-MO2) (5′-GCCACACACTTCCTTACATTCCATAGATTA-3′) were designed complementary to the miRNA guide strand and the Dicer nucleolytic processing sites respectively according.