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First published online 5 October 2005
doi: 10.1242/dev.02020

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Sensitized genetic backgrounds reveal a role for C. elegans FGF EGL-17 as a repellent for migrating CAN neurons

Tinya C. Fleming, Fred W. Wolf^* and Gian Garriga

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204, USA

View larger version (44K): [in a new window]   Fig. 1. slt-1, sax-3, egl-17 and egl-15 mutations enhanced the CAN migration defects of vab-8 and unc-34 mutant. (A) Merged fluorescence and Nomarski images of a newly hatched first larval stage ceh-23::gfp transgenic animal that expresses GFP in the CANs, as well as sensory neurons in the head and tail. Anterior is towards the left and dorsal is upwards. Only the left CAN is visible. Although the ceh-23::gfp transgenic animals shown are used to illustrate the normal positions of the CANs, this transgene sensitizes the background to CAN defects. To avoid this, we scored CAN positions by Nomarski optics in a background lacking this transgene. (B) In a schematic view of the region in which CAN positions were scored, the CAN is shown in a wild-type position and the arrow marks the path of CAN migration. The vertical marks define the positions of the landmark V cell and P cell nuclei, which are also shown in the diagram. (C) For each genotype, the percentage of CANs found in each position is noted along with the total number (n) of CANs scored. The shading of the box reflects the percentage of CANs in that position, with darker shading reflecting higher percentages. Single mutants of the tested guidance cues and receptors did not show CAN migration defects. slt-1(eh15), sax-3(ky200), egl-17(n1377) and several egl-15 mutants enhanced the CAN migration defects of vab-8(gm84) mutants (P<0.01), while unc-6(ev400), unc-40(e1430) and let-756(s2887) did not (P=0.65, P=0.14, P=0.26, respectively). A two-sample z-test on the proportion of CANs found in the most anterior CAN position was used to determine whether double mutants showed significant enhancement of CAN migration defects when compared with vab-8(gm84) single mutants. In particular, vab-8(gm84); slt-1(eh15) and vab-8(gm84); sax-3(ky200) displayed significant enhancement of vab-8(gm84) CAN migration defects with P<0.0001, while vab-8(gm84); egl-15(n484) and vab-8(gm84); egl-15(n1456) strains enhanced with P<0.001. vab-8(gm84); egl-17(n1377), egl-15(n484) mutants displayed CAN migration defects comparable with vab-8(gm84); egl-17(n1377) and vab-8(gm84); egl-15(n484) mutants (P=0.98 and P=0.31, respectively). egl-17(n1377) and egl-15(n1477ts) mutants also enhanced the CAN migration defects (P<0.01) of the unc-34(e951) null allele when using a two-sample z-test on the proportion of CANs found in the two most anterior CAN positions.

View larger version (28K): [in a new window]   Fig. 2. clr-1 suppression of CAN migration defects of vab-8 and unc-34 mutants The CAN positions were scored and are displayed as described in Fig. 1. The temperature-sensitive allele clr-1(e1475ts) significantly suppressed the vab-8(gm84) allele and the null alleles vab-8(e1017) and unc-34(e951). This suppression required EGL-15.

View larger version (28K): [in a new window]   Fig. 3. Mutations in sem-5 but not let-60 enhanced the CAN migration defects of vab-8 mutants. The CAN positions were scored and are displayed as described in Fig. 1. The sem-5 loss-of-function allele n2019 significantly enhanced the CAN migration defects caused by the vab-8(gm84) allele (P<0.01). By contrast, the let-60 hypomorphic allele n2021, the null allele sy101sy127 and the gain-of-function allele n1046 failed to significantly enhance the CAN migration defects of vab-8(gm84) mutants (P=0.70, P=0.96 and P=0.21, respectively).

View larger version (28K): [in a new window]   Fig. 4. Enhancement of CAN migration defects by constitutively active EGL-15 and global egl-17 expression. The CAN positions were scored and are displayed as described in Fig. 1. (A) Global expression of egl-17 from a heat shock promoter significantly enhanced the CAN migration defects of vab-8(gm84). (B) A transgene expressing a constitutively activated EGL-15(neu) enhanced vab-8(gm84) CAN migration defects, while overexpression of wild-type egl-15 did not (P=0.39). Strains containing the transgenes were scored in a soc-2(n1774) background to avoid the lethality caused by constitutive EGL-15 activity.

View larger version (56K): [in a new window]   Fig. 5. egl-17 expression in two anterior cells of the embryo during CAN migration. (A) The CAN positions were scored and are displayed as described in Fig. 1. Expression of egl-17 from its endogenous promoter rescued the enhancement of vab-8(gm84) CAN migration defects by the egl-17(n1377) mutation. vab-8(gm84); egl-17(n1377) mutants with a rescuing egl-17 transgenic array (w/[egl-17+]) displayed CAN migration defects indistinguishable from vab-8(gm84) mutants (P=0.76), while siblings that had lost the array (w/o [egl-17+]) showed defects similar to the original vab-8(gm84); egl-17(n1377) strain (P=0.51). (B) Fluorescence image of a 1.5 fold stage embryo showing expression of an egl-17::gfp transgene (ayIs9) in a cell at the anterior tip of the head. A second cell expressing GFP is located on the other side and is out of the plane of focus. The two arrowheads indicate two cell corpses that were engulfed by the GFP-expressing cell. The two cells appear to be hyp5 hypodermal cells based on their position and morphology during development. (C) Nomarski image of the same embryo in A. The two arrowheads indicate the same cell corpses as shown in A. Scale bar: 10 µm. (D) Schematic lateral view of the embryo at the stage shown in A. The arrow indicates the path of CAN migration. The dark region shows the site of egl-17 expression.

View larger version (45K): [in a new window]   Fig. 6. Ectopic expression of EGL-17 repels CAN cell migrations. (A) Fluorescence images with corresponding schematic diagrams show the GFP expression pattern of the two transgenes, Plim-4::egl-17::gfp and Pmab-5::egl-17::gfp, used to express EGL-17 ectopically. Scale bar: 10 µm. (B) The CAN positions were scored and are displayed as described in Fig. 1. Ectopic expression of EGL-17 in the head from a Plim-4::egl-17::gfp transgene partially rescued vab-8(gm84); egl-17(n1377) CAN migration defects. By contrast, ectopic expression of EGL-17 in the mid-posterior region from a Pmab-5::egl-17::gfp transgene enhanced the CAN migration defects of a vab-8(gm84) background.

View larger version (17K): [in a new window]   Fig. 7. EGL-15 functions cell autonomously in CAN migration. The CAN positions were scored and are displayed as described in Fig. 1. Expression of a Pceh-10::egl-15 transgene rescued the enhancement of the CAN migration defects caused by egl-15(n484) in a vab-8(gm84) background. The ceh-10 promoter drives expression in a small subset of sensory neurons and in the migrating CANs. By contrast, the expression of EGL-15 in hypodermal cells from a Pdpy-7::egl-15 transgene failed to rescue the CAN migration defects of vab-8(gm84); egl-15(n484) (P=0.52).