Maternal syntabulin is required for dorsal axis formation and is a germ plasm component in Xenopus

Highlights

• The kinesin adapter Syntabulin is required for dorsal development in zebrafish.

• Xenopus maternal syntabulin mRNA is vegetally localized in oocytes.

• Xenopus syntabulin mRNA is expressed zygotically in progenitor germ cells.

• Syntabulin maternal depletion results in ventralization in Xenopus.

• Sybu-depleted embryos can be rescued by syntabulin or β-catenin mRNA.

Abstract

In amphibians and teleosts, early embryonic axial development is driven by maternally deposited mRNAs and proteins, called dorsal determinants, which migrate to the presumptive dorsal side of the embryo in a microtubule-dependent manner after fertilization. Syntabulin is an adapter protein that binds to kinesin KIF5B and to the transmembrane protein Syntaxin1. In zebrafish, a mutation in Syntabulin causes complete embryo ventralization. It is unknown whether Syntabulin plays an analogous role during early development of other species, a question addressed here in Xenopus laevis. in situ hybridization of syntabulin mRNA was carried out at different stages of Xenopus development. In oocytes, syntabulin transcripts were localized to the vegetal cortex of large oocytes and the mitochondrial cloud of very young oocytes. We extended the zebrafish data by finding that during cleavage Xenopus syntabulin mRNA localized to the germ plasm and was later expressed in primordial germ cells (PGCs). This new finding suggested a role for Syntabulin during germ cell differentiation. The functional role of maternal syntabulin mRNA was investigated by knock-down with phosphorothioate DNA antisense oligos followed by oocyte transfer. The results showed that syntabulin mRNA depletion caused the complete loss of dorso-anterior axis formation in frog embryos. Consistent with the ventralized phenotype, syntabulin-depleted embryos displayed severe reduction of dorsal markers and ubiquitous transcription of the ventral marker sizzled. Syntabulin was required for the maternal Wnt/β-Catenin signal, since ventralization could be completely rescued by injection of β-catenin (or syntabulin) mRNA. The data suggest an evolutionarily conserved role for Syntabulin, a protein that bridges microtubule motors and membrane vesicles, during dorso-ventral axis formation in the vertebrates.

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.