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First published online 20 July 2005
doi: 10.1242/dev.01947

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The VAB-1 Eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function together during C. elegans embryonic morphogenesis

Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada

View larger version (33K): [in a new window]   Fig. 1. vab-1/Eph is synthetic lethal with sax-3/Robo and displays gene-dose sensitivity. The percentage embryonic lethality of single mutants and double mutant combinations between vab-1, sax-3 and slt-1 is shown. All alleles of vab-1 tested are 100% embryonic lethal with the sax-3(ky123) null. The null allele of vab-1(dx31) displays a dosage sensitivity with sax-3 in that embryos are dead even in the presence of one wild-type copy of vab-1. +/dx31; 0/sax-3 males are also dead. The double heterozygous embryos +/dx31; + or 0/sax-3 includes hermaphrodites and males, and the lethality is greater than 50% (57%), suggesting some non-allelic, non-complementation. By contrast, control crosses + or 0/sax-3 gave 27% embryonic lethality. vab-1; slt-1 does not display synthetic lethality but the phenotypes are enhanced. Error bars represent s.e.m. of at least three broods; n, total number of embryos scored for each genotype.

View larger version (115K): [in a new window]   Fig. 2. sax-3 mutants display defects in cell movements similar to those seen in vab-1 embryos. (A) Cartoon of wild-type C. elegans embryogenesis highlighting some cell movement events during embryogenesis. The gastrulation cleft is closed by lateral neuroblast movements by 200 minutes, and enclosure of the ventral epidermis (red cells) is observed by 325 minutes at 22°C. Lateral (seam cells) are coloured yellow and the dorsal epidermis is coloured orange. Arrows show the direction of cell movements. (B-G) Embryogenesis of N2 (wild type) and various single- and double-mutant combinations. Images are single frames taken from 4D DIC movies of individual embryos. All embryos are shown as ventral views; anterior is to the left. The times are given in minutes relative to the first cleavage, and are shown at the bottom left of each panel. Scale bar: 10 µm. (B) In wild-type (wt) development the ventral gastrulation cleft is small (<15 µm, dotted line). After the cleft is closed, the epidermal cells migrate around the embryo to meet at the ventral midline. The leading cells are marked (arrows). (C) sax-3(ky123) embryos have broader, deeper and more persistent gastrulation clefts because the neuroblasts either fail to move or migrate abnormally. Later when the epidermal cells start to migrate the gastrulation cleft is still open and it is common to see cells `floating' (asterisk) around in the still open gastrulation cleft. The open gastrulation cleft and wandering cells may interfere with the epidermal leading cells (arrows) and posterior pocket cells enclosing the embryo. The embryo usually ruptures at the ventral side during the elongation process. (D) vab-1(dx31) is shown for comparison. (E) Double-mutant vab-1(e2); sax-3(ky123) embryos have cell movement defects that are similar to the phenotypes of either vab-1 or sax-3 single mutants. The gastrulation cleft is usually larger and less defined, as the cells are disorganized (Class I, strongest phenotype). (F) Double-mutant vab-1(dx31); slt-1(eh15) embryos have broader, deeper and more persistent gastrulation clefts. Epidermal cells are highly disorganized and do not migrate to the ventral side, and the embryos usually arrest before ventral enclosure; a phenotype not seen in either of the single mutants. (G) Double mutants efn-4(bx80); sax-3(ky123) are completely inviable and display cell movement defects that are similar to those of the vab-1 ptp-3 and vab-1(dx31); efn-4(bx80) double mutants. The question mark denotes that the embryo was so disorganized that we could not confidently identify whether this cell was the leading cell. ptp-3(op147); sax-3(ky123) also exhibited similar defects (not shown).

View larger version (100K): [in a new window]   Fig. 3. vab-1 and sax-3 display non-allelic non-complementation in axon guidance; DIC (left) and fluorescence (right) images of the head region are shown. (A) +/mIn1GFP `wild-type' adults stained with the vital dye DiI to visualize the amphid head neurons (red). The mIn1GFP marker was used to visualize the pharynx (green). The amphid neuronal cell bodies are close to the terminal bulb (TB) of the pharynx. The amphids send out axons that first migrate ventrally and then dorsally to project and terminate in the nerve ring (arrow), which is posterior to the metacorpus (MC). (B) +/mIn1GFP; sax-3 mutants display a highly penetrant anterior axon displacement. The axons project anteriorly past the metacorpus, arrow. In addition, sax-3 amphid cell bodies are often misplaced more anteriorly. (C) vab-1/mIn1GFP; +/sax-3 double heterozygous animals display axon guidance defects (25%) similar to those of sax-3 homozygous animals.

View larger version (119K): [in a new window]   Fig. 4. Epidermal defects in sax-3(ky123), vab-1(dx31), and vab-1(dx31); slt-1(eh15) mutants. Columns from left to right show ventral, lateral and dorsal views, with the exception of K and L, which are dorsolateral views. In all panels anterior is to the left. All animals carry the ajm-1::GFP (jcIs1) epidermal cell marker. (A-C) Wild-type embryo development. (A) The contralateral epidermal cells meet at the ventral midline and enclose the embryo (arrows point to the leading cell). (B) The seam cells are organized in a lateral row of 10 cells (H0, H1, H2, V1-V6, T). (C) The dorsal epidermal cells fuse to form the hyp 7 syncytium. (D-F) Defects in sax-3 mutants. (D) One of the left leading cells maintained inappropriate contact (double arrow) with both leading cells on the opposite side. The unclosed gastrulation cleft (gc) prevented the pocket cells from connecting at the ventral midline. (E) The positions of V1 and V3 seam cells are ventrally shifted (arrows), leading to abnormal seam cell contacts. (F) One of the dorsal epidermal cells did not fuse with its neighbours and exists as a single cell within the hyp 7 syncytium (arrowhead). (G-I) Defects in vab-1 mutants. (G) The unclosed gastrulation cleft (gc) prevented the leading and pocket cells from enclosing the embryo. (H) V5 is shifted ventrally, allowing a direct contact of V4 with V6. (I) No defects are observed for the dorsal epidermis; however, one of the seam cells (V1) is shifted dorsally (arrow), allowing H2 to contact V2. (J-L) Defects in vab-1; slt-1 double mutants. (J) The ventral epidermis structure is drastically disrupted because of the presence of the enlarged gastrulation cleft (gc). (K) V3 is shifted ventrally (arrow) and the V2 cell makes direct contact to V4. (L) The shape and position of anterior epidermal cells and seam cells are drastically changed. Arrows indicate unidentified (?) seam cells because some appear to fuse to dorsal hyp 7.

View larger version (68K): [in a new window]   Fig. 5. 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.

View larger version (34K): [in a new window]   Fig. 6. The VAB-1 tyrosine kinase domain physically interacts with the juxtamembrane and CC1 region of SAX-3/Robo. (A) Schematic diagrams of the VAB-1 Eph RTK and SAX-3/Robo receptor. VAB-1 Kinase region (669 aa-985 aa) was fused in frame to the GAL-4 DNA-binding domain and used as bait in yeast two-hybrid assays. The SAX-3 intracellular CC1, CC3 and CC2 regions are shown as blue boxes. SAX-3 does not have a recognizable CC0 consensus and CC3 is N terminal to CC2 when compared with other Robo receptors. (B) Deletion constructs of the intracellular portion of SAX-3 were tested for their ability to bind the VAB-1 tyrosine kinase domain. Liquid ß-Galalctosidase units and yeast X-GAL overlay assays indicate the quantity of activation (interaction). The juxtamembrane and CC1 region is necessary and sufficient for the interaction with the VAB-1 kinase region (dashed rectangle). (C) GST `pull-down' experiments confirm that the SAX-3 juxtamembrane and CC1 region (900 aa-1030 aa) is sufficient for the interaction with the VAB-1 intracellular region. MBP-SAX-3 E. coli lysates were incubated with GST or GST-VAB-1. MBP-SAX-3 input (Load), unbound (UB) and bound (B) fractions were analyzed by SDS-PAGE and western blotting using anti-MBP to visualize SAX-3. The SAX-3 interaction is specific to GST-VAB-1 and not GST alone. GST-VAB-1 is also specific for MBP-SAX-3, as it does not bind the MBP degradation products (asterisks) in the Load.

View larger version (16K): [in a new window]   Fig. 7. Model for the function of SAX-3 in C. elegans embryonic development. During embryonic development most cells reach their final position through a combination of local divisions and directed movements. SAX-3/VAB-1 pathways are required for neuroblast movements that result in the closing of the gastrulation cleft. SAX-3/VAB-1 may also be required to position early AB epidermal precursors that will generate the future epidermal seam cells and ventral epidermal cells. Our results are consistent with SAX-3 functioning with VAB-1 in parallel pathways or perhaps through a receptor complex (SAX-3/VAB-1). At the same time SAX-3 functions independently of VAB-1 for epidermal morphogenesis; for example, during the migration of dorsal epidermal cells.