SAX-3 is expressed in a subset of neuroblasts and neurons that express VAB-1. (A) Extrachromosomal arrays carrying SAX-3/Robo in a
sax-3 background have rescuing activity. We scored for rescue of the
sax-3 embryonic lethality and notched-head phenotype (top right). The transgenic lines restore SAX-3 activity in neuroectoblast (F25B3.3::SAX-3), epithelial cells (
ajm-1::SAX-3) or all SAX-3 tissues (SAX-3 mini gene and SAX-3::GFP). By contrast, the
odr-1::RFP transgenic marker had no rescuing activity. Note that the
ajm-1::SAX-3 did not rescue the notched-head phenotype. Complete rescue is not expected, as these transgenic animals carry an extrachromosomal array that is randomly lost during cell divisions, therefore not all animals carry the extrachromosomal array. Error bars indicate s.e.m. from at least three broods; n, total number of embryos scored. Asterisks indicate transgenic animals with significantly less embryonic lethality or notched heads than
sax-3 animals (t-test, P<0.001). Scale bar: 20 μm. (B) SAX-3 is expressed in neurons and in epidermal cells during epidermal ventral enclosure. SAX-3::GFP fluorescence is observed in many neurons (top) and is similar to VAB-1 expression. SAX-3::GFP is also expressed in epidermal cells (bottom). (C) VAB-1 and SAX-3 are co-expressed in some neuroblasts and neurons during development. Three different stages are shown: 100-200 cells (top), pre-ventral enclosure (middle) and comma stage (bottom). Double immunostaining with anti-VAB-1 (red) and anti-GFP (green) was used to visualize the co-localization of VAB-1 and SAX-3. VAB-1 and SAX-3 are expressed in similar cells but their expression patterns are not identical, consistent with these two receptors functioning together in some cells but also having independent roles during development.