Fig 1.
Common posterior Distal Tip Cell (DTC) trajectories in ngat-1(ev821) mutant animals.
Lateral view of the posterior gonad arm in L4 hermaphrodite animals by D.I.C. microscopy. In all panels, anterior is left, dorsal is up. (A) The 2 DTCs are born in ventral mid-body (in position of future vulva marked by triangle) and normally migrate post-embryonically in 3 phases [(1) thru (3)] to shape the developing U-shaped gonad arms. (B) Some ngat-1(ev821) animals grown at 25°C have a normal posterior DTC migration as in panel A, while many have an unc-6 mutant-like phase 2 failure with phases 2 and 3 executed on the ventral side (panel B), while others (panel C) show a partial return to mid-body before migrating dorsally. To the right of panels B and C are the predicted corresponding ventral clear patch (VCP) phenotypes visible by low magnification stereomicroscopy (asterisk marks partial VCP in panel C). Scale bar in panel A (for A-C) is 50 μm.
Fig 2.
DTC navigational and ventral clear patch (VCP) phenotypes of ngat-1(ev821) animals.
The number (#) and percentage (%) of ngat-1(ev821) and GnT1 triple (see below) animals grown at 25°C with a given diagrammed posterior DTC abnormal (i-iv) or wild-type-like (WT = vi) trajectory (deduced from posterior gonad arm shapes) are shown. Diagrams are based on D.I.C. microscopy of gonad arms from a lateral perspective. Predicted corresponding ventral clear patch (VCP) phenotypes are indicated in the right-most column.
Fig 3.
Approximate ts period for the ev821 phase 2 DTC navigation errors.
ngat-1(ev821) animals were grown at 16°C or 25°C then shifted to test temperature (25°C or 16°C, respectively) at various larval stages of development (L1, L2, and L3). Control animals were maintained at 16°C or 25°C test temperature throughout the experiment. Animals were scored by D.I.C. microscopy in the 4th larval stage (L4) or as young adults for phase 2 posterior DTC migration errors. A temperature-sensitive period was readily identified for these defects, which was developmentally much earlier than manifestation of the late L3 stage phase 2 DTC migration defect. Error bars represent z-test based 95% confidence intervals.
Fig 4.
Comparison of galactosyl transferases from C. elegans, D. melanogaster (fly), and humans.
Alignments were made of the most closely-related C. elegans (wormbase), fly (FBgn0027538, FBgn0031495) and human (Q8NCL4, Q10472) galactosyltransferases using CLUSTAL [47]. The ngat-1(ev821) nonsense mutation is indicated by blue triangles and the position of the Leu (or in this case Ile) residue, which specifies N-acetylgalactosamine transfer (vs Tyr or Phe, which do not) [48] is indicated by red triangles. Metal ion (red underline), acceptor (blue underline), and donor (green underline) binding sites as predicted from human galactosyl transferases [45]. Identical residues are shaded black and similar residues are shaded grey.
Fig 5.
Organization of ngat-1 coding sequence and encoded protein.
Boxes represent cDNA exons whose approximate codon size is indicated within. Exon boundaries and the positions of the ev821 point mutation (red) and the ev823 deletion (red) are indicated above the exon boxes. The corresponding protein domain structure [48] is indicated below the exons (Cyto = Cytoplasmic domain, TM = transmembrane domain). Color coding for binding sites is as in Fig 4.
Fig 6.
Common posterior gonad arm phenotypes of the GnT1/Mgat1 triple null mutant.
(A-C) Diagram iv errors of Fig 2 are of various trajectories in ngat-1 and the GnT1 triple mutants following normal phase 1 navigation, three of which are shown here by D.I.C. microscopy. In these, following a normal phase 1, the posterior DTC changes direction often and seemingly randomly, but rarely away from mid-body, although the distal arm in these cases does not return to the mid-body region. Panel C shows a posterior gonad arm like that of diagram ii. The return toward mid-body on the ventral side is usually shorter in the GnT1 triple compared to the ngat-1(ev821) mutant. Premature phase 1 ventral-to-dorsal migrations were observed for 7 anterior DTCs of 130 GnT1 triple mutant animals examined. Scale bar in panel A (for A-C) is 50 μm.
Fig 7.
Temperature-sensitive period for GnT1 triple mutant phase 2 DTC migration errors.
A temperature shift experiment similar to that of Fig 3, but limited to shifting only L1s (based on the Fig 3 ev821 results) was performed for the GnT1 triple null mutant scored for all 3 kinds of VCPs by low magnification stereomicroscopy. Visible VCPs are not as frequent as phase 2 defects scored by D.I.C. in the GnT1 triple mutant, probably because the distal arm return along the proximal arm is typically much shorter (creating a much smaller VCP) in the GnT1 triple than in ngat-1(ev821). Neither the ngat-1(ev821) nor the GnT1 triple null mutant can prevent, reverse, or compensate a phase 2 DTC migration failure induced by embryonic growth at 25°C if shifted back to 16°C during the first larval (L1) phase, whereas embryonic growth at 16°C leads to largely normal DTC migration that cannot be reversed by a shift to 25°C during the first larval (L1) stage.
Fig 8.
ngat-1p::gfp transcriptional reporter expression pattern.
A chromosomally integrated ngat-1p::gfp transcriptional reporter strain was made as described in Materials and methods. A dpy-7p::mCherry transcriptional reporter was injected into the ngat-1::gfp integrated line to create a non-integrated marker for epidermal cells (see Materials and methods), which may not mark all epidermal cells in a given animal because of potential mitotic loss of the extrachromosomal array. (A-B) merge of gfp and mCherry signals showing presumptive mCherry-expressing epidermal cells (thick arrows) that co-express gfp (as indicated by orange tinge) plus presumptive muscle cells (thin arrows), and presumptive intestinal cells (star) that express only ngat-1p::gfp in a comma stage embryo. (C-E) The ngat-1p::gfp reporter is expressed in body wall muscles (arrows in C and E) and in lateral epidermis (asterisks in D and E) in all post-embryonic larval stages. Some unidentified elongated cells in the head also express gfp and at least one (arrowhead) also expresses mCherry. (Dark oval-shaped areas in the lateral epidermis are lateral seam cells). rf = red fluorescence channel; gf = green fluorescence channel. Scale bar in panel A (for A and B) is 12.5 μm. Scale bar in panel C (for C-E) is 100 μm.