Wnt/β-catenin signaling is essential for the initiation of dorsal-ventral patterning during vertebrate embryogenesis. Maternal β-catenin accumulates in dorsal marginal nuclei during cleavage stages, but its critical target genes essential for dorsalization are silent until mid-blastula transition (MBT). Here, we find that zebrafish net1, a guanine nucleotide exchange factor, is specifically expressed in dorsal marginal blastomeres after MBT, and acts as a zygotic factor to promote the specification of dorsal cell fates. Loss-and gain-of-function experiments show that the GEF activity of Net1 is required for the activation of Wnt/β-catenin signaling in zebrafish embryos and mammalian cells. Net1 dissociates and activates PAK1 dimers, and PAK1 kinase activation causes phosphorylation of S675 of β-catenin after MBT, which ultimately leads to the transcription of downstream target genes. In summary, our results reveal that Net1-regulated β-catenin activation plays a crucial role in the dorsal axis formation during zebrafish development.
Many differential gene expression analyses are conducted with an inadequate number of biological replicates. We describe an easy and effective RNA-seq approach using molecular barcoding to enable profiling of a large number of replicates simultaneously. This approach significantly improves the performance of differential gene expression analysis. Using this approach in medaka (Oryzias latipes), we discover novel genes with sexually dimorphic expression and genes necessary for germ cell development. Our results also demonstrate why the common practice of using only three replicates in differential gene expression analysis should be abandoned.
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