GSK-3 Activity Is Critical for the Orientation of the Cortical Microtubules and the Dorsoventral Axis Determination in Zebrafish Embryos

Abstract

The formation of dorsal-ventral (D–V) axis is the earliest event that breaks the radial symmetry and determines the bilateral body plan of a vertebrate embryo, however, the maternal control of this process is not fully understood. Here, we discovered a new dorsalizing window of acute lithium treatment, which covers only less than 10 minutes after fertilization. Lithium treatment in this window was not able to reverse the ventralized phenotype in tokkeabi (tkk) mutant embryos, and its dorsalizing activity on wild-type embryos was inhibited by nocodazole co-treatment. These evidences indicate that the underlying mechanism is independent of a direct activation of Wnt/β-catenin signaling, but depends on the upstream level of the microtubule mediated dorsal determinant transport. In order to identify the target of lithium in this newly discovered sensitive window, GSK-3 inhibitor IX as well as the IMPase inhibitor L690, 330 treatments were performed. We found that only GSK-3 inhibitor IX treatment mimicked the lithium treatment in the dorsalizing activity. Further study showed that the parallel pattern of cortical microtubules in the vegetal pole region and the directed migration of the Wnt8a mRNA were randomized by either lithium or GSK-3 inhibitor IX treatment. These results thus revealed an early and critical role of GSK-3 activity that regulates the orientation of the cortical microtubules and the directed transport of the dorsal determinants in zebrafish embryos.

Fixation/Permeabilization: New Alternative Procedure for Immunofluorescence and mRNA In Situ Hybridization of Vertebrate and Invertebrate Embryos

“…D: Control showing the expression of gsc mRNA in cells of the embryonic shield (es) using the Thisse and Thisse () protocol. Differences between the quality of the in situ hybridizations shown in C and D are confirmed when we compare our results with those obtained by others with a similar method in the same embryonic material (see Shao et al, ). E: Expression of brul mRNA in the lens (arrowhead).…”

“…Similar gastrulae, that we stained for gsc mRNA using a conventional procedure (Thisse and Thisse, 2008), revealed poorer cell preservation and less precise localization of the hybridization signal (compare Figs. 10C and D) (see also Shao et al, 2012).…”

Tumor Suppressor Lzap Suppresses Wnt/β-Catenin Signaling to Promote Zebrafish Embryonic Ventral Cell Fates via the Suppression of Inhibitory Phosphorylation of Glycogen Synthase Kinase 3

“…Indeed, a recent study has shown that Sfrp1 and Frzb are two maternal Wnt inhibitors that limit the spatial context of Wnt proteins in dorsal cells (3). In addition, embryos treated with inhibitors that inactivate GSK3 (required for ␤-catenin degradation) from the 32-cell stage to the sphere stage also result in dorsalized phenotypes (14), indicating that GSK3 activity is promoted in ventral cells to ensure ventral cell fate. It is unclear, however, how GSK3 activity in ventral cells is controlled during early embryogenesis.…”

“…In vertebrates, two genes encode GSK3 (GSK3␣ and GSK3␤) and Axin (Axin1 and Axin2). GSK3␤ is known to be involved in axis formation (14), whereas Axin2 is a direct target of Wnt/␤-catenin signaling and acts as a feedback negative regulator for the same pathway (15). In zebrafish, two genes code for ␤-catenin.…”

Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish

“…The dorsal determinants (DDs) model suggests an intimate relationship between animal-vegetal polarity and the DV axis, because dorsalising signals are enriched in the vegetal pole of the oocyte. Triggered by fertilisation and controlled by microtubules, DDs are asymmetrically transported, from vegetal pole to the prospective dorsal side in zebrafish (Abrams and Mullins, 2009;Mei et al, 2009;Nojima et al, 2010;Langdon and Mullins, 2011;Shao et al, 2012;Tran et al, 2012;Ge et al, 2014) and in Xenopus (Cuykendall and Houston, 2009;Slack, 2014;Carron and Shi, 2016). Several candidate DDs, including Dishevelled, GBP, and Wnt11 in Xenopus (Miller et al, 1999;Weaver et al, 2003;Tao et al, 2005), and Wnt8a in zebrafish (Lu et al, 2011), were thought to be required for dorsal axis formation.…”