The Molecular Mechanism of Early Development
A central question in developmental biology is how a ball of cells patterns itself into a three layered organism with recognizable axes. We have approached this question by carrying out loss of function experiments targeting the earliest phase of gene regulation in development, the time that is controlled by transcription factors and signaling molecules inherited from the mother in the cytoplasm of the egg. In particular, our work has shown the importance of the Wnt signaling pathway, the fact that it is essential to establish the dorsal/ventral axis of the embryo by regulating the expression of genes expressed in the Spemann Organizer region.
The most surprising recent finding is that this particular Wnt pathway relies on two Wnt signaling inputs, involving both Wnt5a and Wnt11, and that these two Wnts interact with each other. This work was published in Development in 2008. More recently we identified the mechanism of their interaction, showing that this relied on an unusual post-translational modification, the tyrosine sulfation of two residues in the C terminal region of each Wnt. We showed that the tyrosyl sulfotranferase enzyme, TPST1, is the catalyst for this sulfation event. This work was published in Current Biology in 2009.
Figure 1. Wnt11/Wnt5a biological interaction. a. LHS: the phenotype of tailbud stage embryos derived from sibling control (Uninj.), Wnt11 depleted (blue, 3ng antisense oligo), Wnt5a depleted (red, 3ng antisense oligo) or Wnt11/5a depleted (3ng Wnt11+3ng Wnt5a antisense oligo, purple). b. Upper panel: real time RTPCR showing the relative expression of maternal Wnt11 and Wnt5a mRNA in matured uninjected oocytes, and oocytes injected with 6ng of Wnt11 or Wnt5a antisense oligos, or with 3ng of both oligos. Lower panel: real time RT-PCR of late blastula stage embryos, siblings to those shown above, analyzing the relative expression levels of the mRNAs of the maternal canonical Wnt pathway target genes Xnr3 and Siamois.
Figure 2. Effects of tyrosyl protein sulfotransferase-1 (TPST-1) depletion. a. Clustal alignment of partial protein sequences of Wnt family members showing the conserved potential sites of sulfation (black) and glycosylation (blue).b Immunoprecipitation experiment from lysates of wild type oocytes and sibling TPST-1 depleted(1KD) oocytes injected with Wnt11-HA and Wnt5a-myc mRNAs. Wnt11-HA is co-immunoprecipitated with Wnt5a-myc in lysates from wild type oocytes, but is much reduced in TPST-1 depleted oocytes (*). Western blots of the input lysates (Input) show the expression levels of the Wnt5a-myc and Wnt11-HA proteins.
