Maternal syntabulin is required for dorsal axis formation and is a germ plasm component in Xenopus
Introduction
A fundamental question in developmental biology is how embryonic body axes are established from an initially symmetrical egg. This developmental event has been extensively studied in Amphibians, particularly in the frog Xenopus laevis, in which the dorso-ventral (D-V) polarity is established soon after fertilization. Sperm entry triggers a series of cytoplasmic rearrangements, known as cortical rotation, which occur before the end of the first cleavage (reviewed in Gerhart et al., 1989, Houston, 2012, Weaver and Kimelman, 2004). During cortical rotation, the outer or cortical vegetal cytoplasm rotates about 30° relative to the inner core cytoplasm in the opposite direction to the sperm entry point, towards the presumptive dorsal side. Externally, this can be followed by the formation of a less pigmented dorsal crescent. Cortical rotation requires the polymerization of microtubules associated to the cortex of the egg, forming parallel bundles with their plus ends oriented away from the sperm entry point (Gerhart et al., 1989, Houliston and Elinson, 1991). Experimental inhibition of cortical microtubule polymerization by nocodazole or ultraviolet (UV) irradiation prevents cortical rotation and ventralizes the frog embryo, causing reduced or missing dorsoanterior structures and expansion of ventral tissues (Scharf and Gerhart, 1983, Elinson and Rowning, 1988).
Cortical rotation coincides with the translocation of vegetally deposited mRNAs, proteins and organelles called maternal determinants, which are transported by kinesin motor proteins towards the plus ends of the cortical microtubules, causing their relocation to the prospective embryonic dorsal side (Gerhart et al., 1989, De Robertis et al., 2000, Weaver and Kimelman, 2004, Houston, 2012). Dorsal determinants are endowed with dorsalizing activity, as shown by their ability to rescue the primary dorsal axis in UV-ventralized embryos and to induce a secondary axis when transplanted into ventral blastomeres of wild type embryos (Holowacz and Elinson, 1993, Kageura, 1997, Marikawa et al., 1997). Thus, the translocation of dorsalizing factors from the vegetal pole to the dorsal side establishes the site where the Spemann Organizer will form at later stages of development (De Robertis et al., 2000, Houston, 2012).
The molecular nature of dorsal determinants remains one of the big mysteries in developmental biology. However, it is clear that the effects of dorsal determinants is to activate the canonical Wnt pathway on the dorsal side of the early embryo, leading to stabilization of the transcriptional co-activator β-Catenin which translocates into the nuclei of dorsal cells (Schneider et al., 1996, Larabell et al., 1997). Several components of the Wnt/β-Catenin signaling pathway have been found to play essential roles in the establishment of Xenopus D-V axis. These include the Wnt ligands Wnt11 and Wnt5A (Tao et al., 2005, Cha et al., 2008), the receptors Frizzled7 (Sumanas et al., 2000) and Lrp6 (Kofron et al., 2007), and cytoplasmic proteins such as Disheveled (Dvl) (Sokol et al., 1995, Miller et al., 1999), Glycogen Synthase Kinase 3 (GSK3)-Binding Protein (GBP) (Yost et al., 1998, Dominguez and Green, 2000) and β-Catenin (Heasman et al., 1994, Heasman et al., 2000). Interestingly, Green Fluorescent Protein (GFP)-tagged versions of both Dvl and GBP proteins were observed to translocate to the prospective dorsal side during cortical rotation, in association to vesicle-like organelles (Rowning et al., 1997, Miller et al., 1999, Weaver et al., 2003).
Despite the realization of the pivotal role of early Wnt signaling in establishing the initial D-V axis in the frog embryo, a mechanistic link connecting dorsal determinant translocation to Wnt signaling activation is still missing. It has recently been found that Wnt signaling requires the endosomal membrane trafficking machinery in cultured cells and Xenopus animal caps (Taelman et al., 2010). When Wnt ligand binds to its receptors, the complex is internalized carrying with it Glycogen Synthase Kinase 3 (GSK3), which becomes sequestered from the cytosol into multivesicular bodies, resulting in the stabilization of proteins such as β-Catenin (Taelman et al., 2010, Dobrowolski and De Robertis, 2011). In addition to GSK3, other components of the activated LRP6 receptor complex such as Dvl-2, Axin and phospho-β-Catenin are also sequestered inside MVBs (Vinyoles et al., 2014). Given that Dvl-containing organelles are transported dorsally (Miller et al., 1999) and the connection between Wnt signaling and endosomal membrane trafficking, proteins that may link membrane vesicles to the microtubule machinery are of great interest.
A new player in the formation of the dorsal axis has been identified by the Hibi laboratory while studying a zebrafish maternal-effect mutation called tokkaebi (tkk) (Nojima et al., 2004). Tkk mutant embryos exhibit a severely ventralized phenotype, lacking all dorso-anterior tissues, and accumulated less β-Catenin in the nuclei of dorsal cells. Importantly, the ventralized phenotype was rescued by overexpression of components of the Wnt pathway such as β-Catenin and Dvl3 (Nojima et al., 2004). The tkk mutation was mapped to the gene encoding for Syntabulin, a Kinesin I linker protein (Nojima et al., 2010). Kinesins are microtubule motors that transport cargo, including membrane vesicles, to the plus-end of microtubules. Interestingly, Syntabulin has also been identified as a protein that binds to the transmembrane protein Syntaxin1 (Su et al., 2004). In neurons, Syntabulin binds to the Kinesin family member 5B (KIF-5B), connecting it to the microtubules, and to Syntaxin1-containing vesicles (Cai et al., 2005, Cai et al., 2007). In neurons, depletion of Syntabulin blocks transport of presynaptic precursor vesicles and mitochondria along the axon to the synaptic terminal, revealing its requirement for the formation of functional synapses (Bury and Sabo, 2011, Cai et al., 2005, Cai et al., 2007, Ma et al., 2009).
Although the precise molecular mechanism of how zebrafish Syntabulin regulates dorsal determination is still unknown, its mRNA has been found to be localized to the vegetal pole of the egg. After fertilization, a Syntabulin antibody showed that this protein translocated from the vegetal pole to the prospective dorsal side of zebrafish zygotes in a microtubule-dependent manner (Nojima et al., 2010). These observations suggested that Syntabulin might be a protein essential for the transport of putative determinants that activate the dorsalizing Wnt signal.
In the present study, we investigated the expression and biological function of the Xenopus syntabulin homolog. Syntabulin transcripts were found to be associated with the mitochondrial cloud of early stage I oocytes and then localized to the vegetal cortex of fully grown oocytes, as is the case in zebrafish. In addition, we found that syntabulin mRNA was associated with germ plasm islands during cleavage, and was expressed in primordial germ cells (PGCs) at later stages. The association between syntabulin and germ plasm/PGCs had not been reported previously. Antisense DNA oligo-mediated knockdown of maternal syntabulin caused radially symmetrical ventralization of frog embryos, with complete loss of dorso-anterior structures, showing a striking similarity to tkk mutant embryos. Notably, syntabulin-depleted embryos could be rescued not only by syntabulin mRNA but also by injection of β-catenin mRNA, reinforcing the idea that maternally stored transcripts of syntabulin are required for the early Wnt signal which triggers dorsal axis formation.
Section snippets
Xenopus syntabulin cloning and antisense oligos
A commercially available full-length Xenopus syntabulin clone (clone ID BC079762, Source BioScience) was identified. For maternal rescue experiments this clone, which contains the 5′ and 3′ untranslated (UTR) sequences, was subcloned into pCS2+ between the ClaI and StuI sites (pCS2-syntabulin-UTR+), and mRNA synthesized with SP6 polymerase after linearization with NotI. For probe preparation the coding sequence was amplified by PCR and subcloned into pCS2+, between the ClaI and XhoI restriction
Xenopus syntabulin is a conserved maternal gene localized to the vegetal cortex
The motor linker protein Syntabulin is characterized by an amino-terminal kinesin binding domain (KBD) and a central syntaxin binding domain (SBD) (Cai et al., 2005, Cai et al., 2007, Su et al., 2004) (Fig. 1A), which enables it to connect syntaxin1-containing vesicle cargoes to kinesin motor proteins for microtubule-dependent transport. Xenopus laevis Syntabulin protein shares an overall high degree of sequence homology with other vertebrate orthologues, such as human and zebrafish (Fig. 1B).
Discussion
Two main findings are reported here. First, Xenopus syntabulin mRNA was not only localized to the vegetal pole of the oocyte but also to the germ plasm of early embryos. Second, maternal depletion of syntabulin mRNA at the very end of oogenesis was sufficient to eliminate dorsal axis formation by the early Wnt/β-Catenin signal. The role of Syntabulin in the establishment of D-V axis was discovered through zebrafish genetics (Nojima et al., 2004). Analysis of the tkk recessive mutant revealed an
Author disclosure statement
The authors declare that there are no conflicting financial interests.
Acknowledgments
We thank Douglas W. Houston for valuable suggestions that helped improve the host transfer experiments, and R. Moon for materials. We thank members of the De Robertis Lab for discussions and critical reading of the manuscript. G.C. is a Howard Hughes Medical Institute Associate. This work was made possible by support from NIH grant HD21502-26, the Norman Sprague Endowment, and the Howard Hughes Medical Institute, of which E.M.D.R. is an investigator.
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