The guanine nucleotide exchange factor Net1 facilitates the specification of dorsal cell fates in zebrafish embryos by promoting maternal β-catenin activation

Abstract

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.

A GEF activity-independent function for nuclear Net1 in Nodal signal transduction and mesendoderm formation

“…Nuclear Net1 also activates RhoA and RhoB in response to DNA damage-associated stimulation (Dubash et al, 2011;Srougi and Burridge, 2011). Furthermore, our previous study uncovered that both nuclear and cytoplasmic Net1 enhance β-catenin S675 phosphorylation in a GEF activity-dependent manner (Wei et al, 2017). These observations raise interesting questions concerning the developmental functions of nuclear Net1, and suggest the need for further investigation.…”

“…1A) (Wei et al, 2017), suggesting that net1 might take part in mesendoderm formation. We previously generated two zebrafish null mutant lines named net1Δ20 and net1Δ53, in which an integrated compensatory network is activated to buffer against the loss of net1 (Wei et al, 2017).…”

“…1A) (Wei et al, 2017), suggesting that net1 might take part in mesendoderm formation. We previously generated two zebrafish null mutant lines named net1Δ20 and net1Δ53, in which an integrated compensatory network is activated to buffer against the loss of net1 (Wei et al, 2017). Therefore, we disturbed net1 expression in embryos by introducing a splice-blocking morpholino (net1 MO1) whose efficiency and lack of off-target effects had been validated (Wei et al, 2017).…”

“…TGF-β-and Nodal-regulated genes usually participate in feedback modulation of signal transduction (Liu et al, 2016;Nicklas and Saiz, 2013;Stroschein et al, 1999). The expression of zebrafish net1 in the blastoderm margin, where mesendoderm precursors are located, is regulated by Nodal signaling (Liu et al, 2011;Wei et al, 2017). However, whether Net1 is involved in Nodal signal regulation and mesendoderm induction remains unknown.…”

“…In addition, this gene is expressed in a dorsoventral-gradient in the blastoderm margin at the onset of gastrulation and has important functions during early embryonic development (Liu et al, 2011). Net1-mediated RhoA activation is required for gastrulation movements during embryonic development of Xenopus and chicken (Miyakoshi et al, 2004;Nakaya et al, 2008), and, in zebrafish (Danio rerio), Net1 and its GEF activity are indispensable for dorsal cell fate specification through promotion of maternal β-catenin activation (Wei et al, 2017). TGF-β-and Nodal-regulated genes usually participate in feedback modulation of signal transduction (Liu et al, 2016;Nicklas and Saiz, 2013;Stroschein et al, 1999).…”