The phenotypic spectrum in Dact1
mutant mice is reminiscent of a similar spectrum of posterior malformations in humans34
. Etiologies previously proposed to explain all or part of this spectrum include vascular-steal35
, persistence of the embryonic cloaca36
, and defects in posterior mesoderm formation37
. Our findings suggest that this malformation spectrum may first originate in disturbed morphogenetic movements at the PS in the early embryo.
Several morphogenetic processes take place in close spatial and temporal proximity in the posterior embryo, each of which is likely to require dynamic regulation and polarized activities of Vangl2, the PCP pathway, and adhesive proteins. The first of these to occur is EMT at the PS, where both Dact1 and Vangl2 are expressed but where neither protein has a prior established role. Our data suggest that defective EMT is the primary defect in Dact1 mutants. Perhaps all other disrupted morphogenesis in these mutants occurs secondarily, for example because of decreased generation of mesoderm and endoderm precursors via EMT. Nonetheless we cannot rule out additional requirements for Dact1 alongside Vangl2 in spatially and temporally adjacent morphogenetic events, including CE movements that elongate and narrow axial mesoderm, mesenchymal-epithelial transition of endoderm precursors, or polarity and cell movements that fold and extend the hindgut.
Wnt/β-catenin signaling and E-cadherin negatively regulate each other at the PS, such that high E-cadherin reduces Wnt/β-catenin signal transduction in cells undergoing EMT38
, and Wnt/β-catenin signaling reciprocally negatively regulates E-cadherin transcription39
. We have observed no statistically significant alterations in Wnt/β-catenin signaling or in levels of E-cadherin mRNA in early Dact1
mutant embryos. Instead the E-cadherin protein is abnormally redistributed at early stages, whereas Wnt/β-catenin signaling reductions occur only much later in morphologically disrupted tail buds where they might contribute to somitogenesis failure specific to late-forming posterior segments (Supplementary Fig 6c-h
. In any case, the rescue of all embryonic Dact1
mutant phenotypes by concurrent mutation of Vangl2
powerfully demonstrates that any E-cadherin or Wnt/β-catenin effects contributing to these phenotypes must occur downstream of Vangl2-dependent events.
We have discovered that a developmentally crucial function of Dact1 is regulation of Vangl2 at the PS. Morphogenetic abnormalities in the PS region of Dact1
mutant embryos, while partly resembling those caused by PCP pathway reduction4
, are unique in that they are associated with decreased phosphorylation of the myosin binding subunit of myosin phosphatase (Mypt1), but increased JNK activity. This agrees with evidence that modulations in Rho-and Jun-kinase activities reflect separate pathways downstream of PCP4
, and that either abnormal gain or loss of upstream PCP components can cause similar disruptions in cell polarity, cytoskeletal dynamics, and CE movements41-44
. Interestingly, other PCP phenotypes, such as inner ear and cardiac malformations18,45
, are not evident in Dact1
mutants (M. Montcouquiol, D.A.F., B.N.R.C., data not shown).
We propose that complex formation between the Dact1 and Vangl2 proteins is a crucial step in a novel Vangl2 regulatory pathway operating during EMT at the PS. Candidate cellular processes that might be involved include trafficking of Vangl2 to the membrane, endocytosis, subcellular sequestration, or degradation. This will be clarified by identifying additional components of this pathway through further biochemical, cellular, embryonic, and genetic experimentation.