Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 9000 Rockville Pike, Building 35, Room 1B 205, Bethesda, MD 20892, USA.
Loss of the zebrafish Nodal-related protein Squint causes a spectrum of phenotypes including cyclopia and midline bifurcations (MB). Here we examine MBs and their relation to cyclopia in maternal-zygotic squint (MZsqt) mutants. There is a concordance of MB with cyclopia in MZsqt embryos. Heat treatment and depletion of Hsp90a are "common" risk factors, each of which increases the incidence of both phenotypes. Midline identity is specified on both sides of MBs, and deep-layer cells are initially lacking within bifurcations, whereas enveloping layer cells are intact. Bifurcations do not appear until the completion of gastrulation and are preceded by gaps in the expression of wnt5b, an essential regulator of dorsal convergence. The incidence of early MBs and wnt5b expression defects in heated MZsqt embryos is high, but there is also substantial recovery. Wnt5b depletion increases the incidence of MB, but not cyclopia, and as such Wnt5b is a "unique" risk factor for MB. Reciprocally, depletion of Wnt11 or Hsp90b increases cyclopia only. In summary, we find that MB arises after gastrulation in regions that fail to express wnt5b, and we show that two complex dysmorphologies - MB and cyclopia - can be promoted by either common or unique risk factors.