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Supplementary data 1. Abnormality of AIY interneurons in sax-7 mutants.
(A) The diagram of AFD thermosensory neurons and AIY interneurons. (B) The
photograph of AFD and AIY labeled with GFP. AIY cell bodies are localized on
ventral side in wild type. (C-F) Variable abnormalities of AIY in sax-7 mutants. (C)
Normally localized AIY. (D) Anteriorly mislocalized AIY.
(E) One of AIY is
mislocalized on dorsal side. (F) Posteriorly mislocalized AIY.
White arrows indicate
cell bodies of AFD and yellow arrows indicate these of AIY. An arrow head indicates
the nerve ring. Scale bar is 20um
(G) AIY cell body phenotypes were scored in sax-7 mutants. n indicates number of
AIY neurons observed.
Supplementary data 2.
Identification of the molecular lesions of sax-7 mutants.
(A) Genomic organization of the SAX-7 on cosmid clone C18F3. Exons are shown in
box. Cosmid clone C18F3 covers the whole region of sax-7 gene. The length of
C18F3 consist of 32792 bases. (B) The display of start and end point of each exon
relative to cosmid C18F3. (C) Amino acid sequences of SAX-7. The predicted signal
sequence is indicated with green, Ig-like domains are indicated with orange, FNIII
repeats are indicated with blue, C-terminal PDZ binding motif is indicated with yellow,
and the transmembrane domain is boxed. (D) The detail of molecular lesions of sax-7
alleles.
Supplementary data 3.
Quantitative analysis of cell body and nerve ring positions
relative to the pharynx.
(A) The diagram of AFD, AWB, and AWC neurons in head region of C. elegans. AFD
cell body is indicated by magenta. AWC cell body located on ventral side and AWB cell
body located on dorsal side are indicated by green. We defined the distance of posterior
half part of pharynx as Dp (Distance of pharynx). Dp is the distance from the grinder
in terminal bulb to the swell of anterior bulb as shown in diagram. We defined Dnr
(Distance of nerve ring ), DAFD, DAWB, and DAWC (Distance of AFD, AWB, and AWC,
respectively) like as Dp. Dnr is the distance from the grinder in terminal bulb to the
point where nerve ring axons meet the midline. DAFD, DAWB, and DAWC are the
distances from the grinder in terminal bulb to the center of each cell body.
We scored
these parameters both in wild type and sax-7(nj48) mutants at several developmental
stages and compared the spatial relationship of cell body and nerve ring positions
relative to the pharynx ( Time 0 is when eggs were laid. After cultivation at 20 degree
for 16, 33, 48, 72, 92, 110 hrs, these parameters were scored ). We directory scored
these parameters under Zeiss Axioplan2 with micrometer and calibrated to um.
(B) The graphs show the developmental transition of Dp, Dnr, D AFD, DAWB, and DAWC
in wild type and sax-7(nj48). The X axis indicates the developmental time (hr) and Y
axis indicates the distance from the grinder in terminal bulb(um). Time 0 is when eggs
were laid.
After cultivation at 20 degree for 16, 33, 48, 72, 92, 110 hrs, these
parameters were scored. More than 40 animals were scored at each developmental stage.
The score of Dp is indistinguishable between wild type and sax-7 mutants. This data
suggests that the pharynx structure is invariable in sax-7 mutants and the pharynx is a
stable land marker to examine the positions of nerve rings and cell bodies.
At the first
larval stage (16hr), the positions of nerve ring and cell bodies of AFD, AWB, and AWC
in sax-7(nj48) mutants were indistinguishable from those of wild type. However, the
positions of these cell bodies are mislocalized anterior to the nerve ring after the second
larval stage. At the adult stage (92 hr), the positions of cell bodies in sax-7(nj48)
mutants are about 10um anteriorly shifted relative to the correctly positioned cell bodies.
We also found that the positions of nerve ring axons are about 10um posteriorly shifted
relative to the correctly positioned nerve ring axons. Not only cell body positions but
also nerve ring positions are abnormal in sax-7 mutants. One explanation for posteriorly
misplacement of nerve ring is that anteriorly misplaced cell bodies physically push the
nerve ring posterior, namely posteriorly misplaced nerve ring is secondary effect of
misplacement of cell bodies. Alternatively, it is possible that SAX-7 has dual functions
for cell body placement and nerve ring placement. We think that the first explanation is
more likely, because some of the AIY interneurons’ cell bodies that are located ventral
side in wild type and are spatially far from the nerve ring are mislocalized dorsally,
anteriorly, or posteriorly in sax-7 mutants(Supplementary data1). If the nerve ring
position was shifted posterior in sax-7 mutants, it is difficult to explain the abnormal
positioning of AIY cell bodies.
(B) The relative quantification of cell body and nerve ring positions. We defined Dnr/Dp,
DAFD/Dp, DAWB/Dp, and DAWC/Dp as relative value of nerve ring and cell body
positions. The X axis indicates the developmental time (hr) and Y axis indicates the
percentage of relative positions.