Development 121, 2655-2666 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
2655
Mosaic analysis of the let-23 gene function in vulval induction of
Caenorhabditis elegans
Makoto Koga and Yasumi Ohshima*
Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan
*Author for correspondence
SUMMARY
The let-23 receptor tyrosine kinase gene is required for
vulval induction and larval survival in the nematode
Caenorhabditis elegans. We carried out genetic mosaic
analyses of the let-23 gene function by using the cloned let23 and ncl-1 genes. The wild-type let-23 gene was required
in a vulval precursor cell to adopt the 1˚ vulval fate in
animals carrying a let-23 vulvaless or lethal chromosomal
mutation. In almost all the animals, vulval precursor cells
adjacent to a 1˚ fate cell were induced to the 2˚ vulval fate
regardless of the let-23 genotypes. These findings indicate
INTRODUCTION
Vulval induction of C. elegans has been studied intensively as
a model system to elucidate the mechanisms by which intercellular interactions specify a pattern of cell fates (Horvitz, 1988;
Sternberg, 1990). Prior to vulval induction, six vulval precursor
cells (VPCs, P3.p - P8.p) locate in the ectoderm along the ventral
midline under the gonad in wild-type hermaphrodites (Sulston
and Horvitz, 1977), and they are considered to have equivalent
developmental potency (Kimble and Hirsh, 1979; Sulston and
White, 1980). During vulval induction, the VPC nearest to the
anchor cell (AC) in the gonad (P6.p) is induced to adopt the 1˚
fate and the adjacent VPCs (P5.p and P7.p) to the 2˚ fate (see
Fig. 5A). From these three VPCs, 22 descendant cells, which
form the vulval tissue, are produced. The other three VPCs(P3.p,
P4.p and P8.p) adopt the hypodermal fate (3˚ fate), and after one
division fuse with the surrounding hypodermal syncytium hyp7
(Sulston and Horvitz, 1977).
In formation of the fate pattern among the VPCs (3˚-3˚-2˚1˚-2˚-3˚), three intercellular interactions are thought to occur.
The major one is an induction signal from the AC. In an animal
in which the AC was ablated by a laser, P5.p-P7.p, which
normally adopt 2˚-1˚-2˚ fates, adopted the 3˚ fate as well as the
other VPCs, suggesting that the AC induces three of the six
VPCs to adopt 1˚ and 2˚ fates (Kimble, 1981). In mutants in
which the location of the AC relative to the VPCs was different
from that of wild type, a new set of VPCs, centered around the
mislocated AC, adopted 2˚-1˚-2˚ fates (Sternberg and Horvitz,
1986; Thomas et al., 1990). In mutants or laser-ablated animals
in which several VPCs were missing, a new set of the
remaining VPCs centered around the AC adopted 2˚-1˚-2˚ fates
that the vulval induction signal from an anchor cell induces
a vulval precursor cell to adopt the 1˚ fate through LET23, and then a 1˚ fate cell induces adjacent cells to adopt
the 2˚ fate, for which LET-23 is not required. Foci of
lethality of the let-23 (mn23) mutation were found in ABal
and ABplp lineages.
Key words: nematode, Caenorhabditis elegans, vulval induction, let23, tyrosine kinase, mosaic analysis, cell signaling
(Sulston and White, 1980; Sternberg and Horvitz, 1986). Furthermore, in a mutant in which only one VPC was produced,
the isolated VPC adopted one of 1˚, 2˚ and 3˚ fates depending
on its distance from the AC (Sternberg and Horvitz, 1986).
Based on these observations, a model involving a graded
anchor cell signal was postulated, namely that an inductive
signal (or signals) secreted from the AC makes a graded positional signal to which each VPC responds to adopt one of the
three fates (Sternberg and Horvitz, 1986).
The second, presumed intercellular signal in vulval
induction is a lateral signal from a 1˚ fate cell, which prevents
the neighboring VPCs from adopting the 1˚ fate. This signal
was presumed since 1˚ fate cells were not observed in positions
adjacent to each other in multivulva (Muv) mutants, such as
lin-15, in which all the VPCs were induced to 1˚ or 2˚ fate in
an AC independent manner (Sternberg, 1988). The lin-12 gene,
which encodes a membrane spanning protein similar to
Drosophila Notch, is known to be involved in this lateral
signaling (Greenwald et al., 1983). lin-12(0) mutants, in which
lin-12 activity is eliminated, have no 2˚ fate cells and additional 1˚ fate cells in place of 2˚ fate cells, while in lin-12(d)
mutants, in which lin-12 activity is elevated and AC is absent,
all the VPCs adopt the 2˚ fate (Greenwald et al., 1983;
Ferguson et al., 1987; Sternberg and Horvitz, 1989). Based on
genetic studies on the interactions among mutations of lin-12,
vulvaless genes (lin-3, lin-2, lin-7 and lin-10) and a multivulva
gene (lin-15), a model based on the combined action of two
intercellular signaling pathways was proposed (Sternberg and
Horvitz, 1989). In this model, the 1˚ fate is induced by a signal
from the AC and the 2˚ fate is induced by a lateral signal, which
is received through LIN-12.
2656 M. Koga and Y. Ohshima
The third intercellular signal is presumed to be inhibitory
and to originate from the surrounding hypodermal syncytium
hyp7, which prevents VPCs from adopting 1˚ or 2˚ fates in the
absence of an inductive signal from the AC. This signal was
revealed by a mutation of lin-15 and its mosaic analysis
(Herman and Hedgecock, 1990). Loss of lin-15 activity causes
a Muv phenotype, and the focus of this gene function is not in
the VPCs or in the AC. lin-15 probably acts in the hypodermis
giving a general inhibitory signal to the VPCs.
Genetic analyses identified more than 20 genes which were
required for vulval induction (Ferguson and Horvitz, 1985), and
molecular cloning revealed that several of those genes encode
proteins homologous to signal transducing molecules in vertebrates. lin-3, let-23, sem-5, let-60, lin-45 and mpk-1/sur-1
encode proteins homologous to TGFα, a receptor tyrosine kinase
(RTK) related to epidermal growth factor (EGF) receptor, an
SH3-SH2-SH3 adapter, Ras, Raf and a mitogen activated protein
kinase (MAP kinase), respectively (Hill and Sternberg, 1992;
Aroian et al., 1990; Clark et al., 1992; Han and Sternberg, 1990;
Han et al., 1993; Lackner et al., 1994; Wu and Han, 1994). Loss
of function or reduced function mutations in these genes, except
mpk-1 (which can only suppress let-60 dominant Muv), cause
all the VPCs to adopt the 3˚ fate (a vulvaless phenotype, Vul).
Elevation of let-60 activity (by a gain of function mutation) or
lin-3 activity (by multi-extragenic copies of the wild-type gene)
causes P3.p, P4.p and P8.p to adopt 1˚ or 2˚ fates as well as P57.p cells (a multivulva or Muv phenotype) (Beitel et al., 1990;
Han et al., 1990; Hill and Sternberg, 1992). Genetic epistasis
experiments suggest that let-23 RTK acts downstream of lin-3
and upstream of let-60 Ras (Ferguson et al., 1987; Han and
Sternberg, 1990), whereas lin-45 Raf and mpk-1 MAPK act
downstream of let-60 (Han et al., 1993; Lackner et al., 1994; Wu
and Han, 1994). These data are consistent with an RTK mediated
signal transduction pathway in vertebrates.
Information on the cellular association of these genes is
expected to shed light on the mechanisms of specification of
vulval fates and on the role of cellular interactions among AC,
VPCs and hyp7. A lin-3::lacZ fusion gene was expressed
specifically in the AC, and Muv phenotypes by multi-extragenic copies of the lin-3 depended mostly on the existence of
the gonad (Hill and Sternberg, 1992). These findings show that
lin-3 acts in the AC. mpk-1 was suggested to act within VPCs
by genetic mosaic analysis (Lackner et al., 1994), but which
VPCs require mpk-1 was not determined. Molecular and
genetic studies suggest that LET-23 is a receptor for the vulval
induction signal from the AC, probably LIN-3 (Aroian et al.,
1990; Hill and Sternberg, 1992). However, the site of LET-23
action is unknown, as is the cellular association of SEM-5,
LET-60 or LIN-45. A key question is, which is the major determinant for 2˚ fate induction, an intermediate inductive signal
from the AC or the lateral signal from a 1˚ fate cell. We determined which cells required the let-23 gene for vulval induction
by examining genetic mosaic animals with respect to let-23.
The results indicate that a 1˚ fate cell is first induced by a signal
from the AC depending on let-23, then 2˚ fate cells are induced
by a 1˚ fate cell independently of let-23.
MATERIALS AND METHODS
General methods for handling nematodes
Methods used for culturing, handling and genetic manipulation of C.
elegans were those described by Brenner (1974). All experiments
were performed at about 20°C. The standard C. elegans cellular and
genetic nomenclature, defined by Sulston and Horvitz (1977) and
Horvitz et al. (1979), respectively, are followed in this paper.
Source strains
The standard wild-type strain N2 and other mutant strains were
obtained from the Caenorhabditis Genetics Center (USA). Below is a
list of alleles used in this work. The source of alleles other than from
Brenner (1974) or the Caenorhabditis Genetics Center are also
indicated. LGII : let-23(sy97), let-23(sy97) unc-4(e120) (Aroian and
Sternberg, 1991), let-23(mn23)unc-4(e120), balancer mnC1[dpy10(e128)unc52(e444)](II) (Herman 1978). LGIII: free duplication
sDp3 (Rosenbluth et al., 1985), dpy-1(e1) ncl-1(e1865) (from C. Kari
and R. Herman).
Generation of transgenic animals
An uncut plasmid or cosmid DNA was injected into hermaphrodite
gonad distal arms (Fire, 1986; Mello et al., 1991), using a MO-202
micromanipulator (Narishige) and an Axiovert-35 inverted microscope (Zeiss). The animals were allowed to recover in M9 buffer and
transferred onto NG plates seeded with E. coli. Transgenic animals
from F1 progeny (identified by their non-Vul phenotype) were cloned
individually and animals that transmitted the genes to their progeny
were selected.
Strain construction
let-23(sy97)/mnC1[dpy-10(e128)unc52(e444)];
dpy-1(e1)
ncl1(e1865) : dpy-1(e1) ncl-1(e1865); sDp3 males were mated with let23(sy97) /mnC1[dpy-10(e128)unc52(e444)] hermaphrodites. Male
cross progeny were backcrossed to let-23/mnC1 hermaphrodites on
four new plates and F1 cross progeny were allowed to self-fertilize on
the same plates. Non-Dpy and non-Vul F2 progeny from the second
cross were picked and placed individually on plates where they were
allowed to self-fertilize. Dpy (dpy-1/dpy-1 and not carrying sDp3) hermaphrodites were selected from broods which segregated dpy-1,
mnC1 and let-23 animals, from which Ncl animals were chosen under
Nomarski optics.
let-23(sy97)/mnC1[dpy-10(e128)unc52(e444)]; dpy-1(e1) ncl1(e1865); sDp3: dpy-1(e1) ncl-1(e1865); sDp3 males were mated
with let-23(sy97) /mnC1[dpy-10(e128)unc52(e444)]; dpy-1(e1) ncl1(e1865) hermaphrodites. Non-Dpy male cross progeny were backcrossed to let-23 /mnC1; dpy-1 ncl-1 hermaphrodites. Non-Dpy and
non-Vul cross progeny from the second cross were placed individually on plates where they were allowed to self-fertilize. Non-Dpy and
non-Vul animals from broods which segregated dpy-1, mnC1 and let23 animals were saved.
let-23(sy97) unc-4(e120); ksEx9[let-23 (+) hsp16-lacZ]: pk7-13.8
plasmid carrying the entire let-23 gene (Aroian et al. 1990) and
pPCZ1 plasmid carrying the lacZ gene under control of the hsp16-1
promoter (Stringham et al. 1992) were coinjected at 50 ng/µl into let23(sy97) unc-4(e120) hermaphrodites. Non-Vul transformant lines
were saved.
let-23(sy97) unc-4(e120); ncl-1(e1865); ksEx9[let-23(+) hsp16lacZ]: let-23(sy97)/mnC1[dpy-10(e128)unc52(e444)]; dpy-1(e1) ncl1(e1865); sDp3 males were mated with let-23(sy97) unc-4(e120);
ksEx9[let-23 (+) hsp16-lacZ] hermaphrodites. Non-Unc cross
progeny were allowed to self-fertilize. Non-Dpy non-Vul Unc animals
from broods which segregated mnC1 and Unc non-Vul animals were
picked, from which Ncl (without sDp3) animals were chosen under
Nomarski optics. Non-Dpy non-Vul Unc progeny from the Ncl
animals were allowed to self-fertilize individually on plates and NonDpy non-Vul Unc animals from broods which did not segregate dpy1 were chosen.
let-23(sy97) unc-4(e120); ncl-1(e1865); ksEx10[let-23(+) ncl1(+)]: let-23 gene (pk7-13.8 plasmid, 50 ng/µl) and ncl-1 gene
(C33C3 cosmid, 150 ng/µl, a gift from David Waring, Fred Hutchin-
Mosaic analysis of let-23 gene function 2657
son Cancer Research Center, USA) were coinjected into let-23(sy97)
unc-4(e120); ncl-1(e1865) hermaphrodites which were obtained as
Unc Vul segregants from let-23(sy97) unc-4(e120); ncl-1(e1865);
ksEx9[let-23(+) hsp16-lacZ]. Non-Vul non-Ncl transformant lines
were saved.
let-23(sy97); ncl-1(e1865); ksEx10[let-23(+) ncl-1(+)]: let23(sy97)/mnC1[dpy-10(e128)unc52(e444)]; dpy-1(e1) ncl-1(e1865);
sDp3 males were mated with let-23(sy97) unc-4(e120); ncl-1(e1865);
ksEx10[let-23(+) ncl-1(+)] hermaphrodites. Non-Unc cross progeny
were allowed to self-fertilize. Non-Unc non-Vul non-Dpy animals
from broods which did not segregate mnC1 were placed individually
on plates. Non-Unc non-Vul non-Dpy animals were saved from
broods which did not segregate dpy-1 or unc-4. The lack of sDp3 was
confirmed by the fact that the animals without ksEx10 were Ncl.
let-23(mn23) unc-4(e120); ncl-1(e1865); ksEx10[let-23(+) ncl1(+)]: let-23(mn23) unc-4(e120)/mnC1[dpy-10(e128)unc52(e444)]
males were mated with let-23(sy97); ncl-1(e1865); ksEx10[let-23(+)
ncl-1(+)] hermaphrodites. F1 progeny were picked and placed individually on plates where they were allowed to self-fertilize. Unc F2
progeny were allowed to self-fertilize. Unc F3 animals showing Ncl
mosaics were selected under Nomarski optics.
Mosaic analysis
Mosaic animals arose spontaneously from a somatic loss of an extrachromosomal array ksEx10[let-23(+) ncl-1(+)] in the progeny from
let-23(sy97) unc-4(e120); ncl-1(e1865); ksEx10 or let-23(mn23)
unc-4(e120); ncl-1(e1865);ksEx10 parents. Using Nomarski optics,
somatic tissues were examined for cells with enlarged nucleoli or Ncl
phenotype (Leung-Hagesteijn et al., 1992; Herman, 1984). When a cell
carried the Ncl phenotype, the somatic genotype of the cell was
assigned as ncl-1, that is, the cell was assumed to have lost ksEx10.
The point of ksEx10 loss was deduced from the genotypes of cells
having the Ncl phenotype, with reference to the invariant cell lineage
of wild-type animals (Sulston et al., 1983; Sulston and Horvitz, 1977)
and by assuming the minimum loss of ksEx10 that is consistent with
the Ncl phenotype pattern in each animal (Herman, 1984). In practice,
first several landmark cells (nuclei) were examined to identify mosaic
animals: they are cells of the pharyngeal muscles m3DL and m3DR
for mosaic animals in which ksEx10 had been lost in MS founder cell
during development (MS mosaics); m3L and m3VL for ABalpa; m3R
and m3VR for ABar; hyp10, hyp9, hyp8, T.appa, B, F, vulval cells
derived from VPCs and neurons derived from Pn.a cells for ABp and
its sublineages to VPCs. If an Ncl cell was observed, other cells related
to the Ncl cell were examined to determine where ksEx10 was lost: for
example, m4, m5, m7, distal tip cells, body wall muscles (derived from
MS, C, D), gonadal anchor cell, pharyngointestinal valve cells, intestinal cells and hyp11 for MS, EMS, C, D, P2 or P1 mosaics; m1, m2,
pharyngeal marginal cells, nerve ring neurons, seam hypodermal cells
for ABalpa, ABar or ABa mosaics; seam hypodermal cells (derived
from H1L, V3 and V5), excretory cell, U and Y for ABp or its sublineages to VPCs mosaics. With this procedure all of ABa, ABalpa,
ABar, ABp, ABpr, ABpl, the other ABpl/r sublineages to VPCs, P1,
P2, EMS and MS mosaics were detected, and some P2, E, C and D
mosaics were also found. The other peripheral mosaics were missed.
Since we were not able to identify individual nerve ring neurons, ABal,
ABalp and ABalpa mosaics could not be distinguished from each
other, and they are represented as ABalpa mosaics. An animal that
showed mosaicism both in ABalpa and ABar was classed as an ABa
mosaic. To avoid misjudgment due to consecutive mosaics of ABalpa
and ABar, another requirement was that many nerve ring neurons were
Ncl, suggesting that ABala and ABalpp had also lost ksEx10.
RESULTS
Outline of the mosaic analysis
To determine where the let-23 gene function is required, we
used a mosaic analysis method similar to that reported recently
(Lackner et al., 1 9 9 4). We made two strains carrying an
extrachromosomal array ksEx10[let-23(+), ncl-1(+)] and
chromosomal mutations ncl-1, unc-4 and either let-23(sy97), a
reduced function vulvaless allele, or let-23(mn23), a null lethal
allele (Aroian and Sternberg, 1991; Herman, 1978). The extrachromosomal array ksEx10[let-23(+), ncl-1(+)] was made by
coinjection of a let-23(+) plasmid and a ncl-1(+) cosmid, a cellautonomous marker gene, by germline transformation. As with
other extrachromosomal arrays, ksEx10 is presumed to be a
mini chromosome-like element consisting of about a hundred
copies of let-23 and ncl-1 DNA clones, which is generated by
repeated homologous recombinations (Mello et al., 1991;
Stinchcomb et al., 1985). This presumption is supported by the
finding that let-23(+) and ncl-1(+) behaved genetically in a
completely linked manner. Also, ksEx10 is slightly unstable
and occasionally lost during a somatic cell division, like other
extrachromosomal arrays.
F1 animals of these two strains at middle L3 to L4 stages
were randomly picked, and screened for mosaic animals in
which a part of, but not all, the somatic cells had enlarged
nucleoli or the Ncl mutant phenotype. At the same time, VPC
descendants were examined for their number and arrangement
to identify the fates of the VPCs; 1˚, 2˚ or 3˚ fates (Fig. 1) as
described previously (Sulston and Horvitz, 1977; Sternberg and
Horvitz, 1986). Table 1 shows the F1 populations we examined.
105 or 179 mosaic animals were detected among the 2053 sy97
or 2082 mn23 animals examined (Table 1). In the sy97 strain,
Table 1. Phenotypes of F1 animal populations
F1 animals
Genotype of parents
let-23(sy97) unc-4;
ncl-1; Ex10[let-23(+), ncl-1(+)]
let-23(mn23) unc-4;
ncl-1; Ex10[let-23(+), ncl-1(+)]
Total number
of worms
examined
2053
2082
All Ncl
Vul
Mosaic
Inv
170
11
93.9%
6.1%
8.8%
0
0
0.0%
105
All non-Ncl
normal
vulva
Vul
1609
91.1%
85
4.8%
1839
96.6%
16
0.8%
5.1%
179
8.6%
shifted
Cov
shifted
Muv
73
0
4.1%
0.0%
86.1%
0
0.0%
0
0.0%
44
3
2.3%
0.2%
91.4%
0
0.0%
1
0.1%
Inv
shifted
Inv
All Ncl and All non-Ncl mean animals in which all the cells examined lacked and retained, respectively, the extrachromosomal array carrying let-23(+) and
ncl-1(+) genes. Mosaic means mosaic animals carrying both types of cells. Vul: vulvaless, Inv: incomplete vulva, Cov: complete vulva, Muv: multivulva.
2658 M. Koga and Y. Ohshima
Fig. 1. Nomarski photomicrographs showing vulval induction in let-23 mosaics. These are all lateral views of a central part of the body at early
L4 stage. Brackets indicate descendants of VPCs and numbers from 4 to 8 correspond to progenitor VPCs from P4.p to P8.p. Open squares
indicate Ncl VPCs. An arrow indicates an Ncl nucleolus of P5.ppp and an arrowhead indicates a non-Ncl nucleolus of P7.paa. All photographs
have the same magnification, and the scale bar represents 20 µm. (A) A normal vulva in a wild-type N2 animal. (B) A vulvaless animal of let23(sy97) unc-4; ncl-1. (C) A non-mosaic animal of let-23(sy97) unc-4; ncl-1; ksEx10[let-23(+) ncl-1(+)], which shows a normal vulva. (D) A
complete vulva in a mosaic animal of let-23(mn23) unc-4; ncl-1; ksEx10[let-23(+) ncl-1(+)]. (E) A shifted complete vulva in a mosaic animal
of let-23(mn23) unc-4; ncl-1; ksEx10. (F) A shifted-Muv in a mosaic animal of let-23(mn23) unc-4; ncl-1; ksEx10.
94% (170/181) of “all Ncl” animals, which probably developed
from zygotes without ksEx10, were Vul (vulvaless) and all the
VPCs adopted the 3˚ fate. The other 6% showed an incomplete
vulva in which one or two P5.p-P7.p adopted 1˚, 2˚ or partial
vulval fate (a cell division pattern partially similar to that of 1˚
or 2˚ fates to produce 3 to 6 cells). In the mn23 strain, no “all
Ncl” animals were observed. Therefore, in these strains, penetrances of let-23(sy97) vulvaless and let-23(mn23) lethal
mutations were 94% and 100%, respectively. Since only 11%
of let-23(sy97) mutants can survive past L2 stage (Aroian and
Mosaic analysis of let-23 gene function 2659
Fig. 2. Mosaic analysis of animals carrying a
chromosomal let-23(sy97) vulvaless mutation, showing
that the wild-type let-23 gene is required in the ABp
lineage for normal vulval induction. Relevant cells and
their lineages are shown together with mosaic animals
represented by the symbols. Each symbol represents a
phenotype of the vulva; filled circle, normal vulva; filled
square, vulvaless (Vul); filled triangle, incomplete vulva
(Inv); open circle, shifted-complete vulva; open triangle,
shifted-incomplete vulva; open diamond, shiftedmultivulva. The position of a symbol in the cell lineage
shows where the extrachromosomal array, ksEx10, has been lost during development of that animal. A question mark indicates a lineage in
which mosaics were not identified because of difficulties in cell identification or in discrimination between Ncl and non-Ncl phenotypes. Some
events in the cells indicated by an asterisk may have been missed because we did not always observe these cell lineages intensively. A number
beside a symbol indicates a particular consecutive mosaic animal.
Sternberg, 1991), it is estimated that about 1500 (181/0.11-181)
“all Ncl” animals of the strain carrying sy97 were dead and
therefore uncounted. Similarly, about 1800 “all Ncl” animals of
the mn23 strain were dead and uncounted, assuming that the
probability of production of zygotes without ksEx10 was the
same as that in the sy97 strain. In fact, we observed many small
dead larvae, roughly corresponding to the above numbers. All
of the “all non-Ncl” animals, which did not lose ksEx10 in any
cell lineages examined, but may include mosaics in peripheral
lineages, are ideally expected to have a normal vulva. However,
a small number of “all non-Ncl” animals (8.9% and 3.4% in the
sy97 and mn23 strains, respectively) were vulvaless (Vul), had
an incomplete vulva (Inv), a shifted incomplete vulva, or a
shifted multivulva (shifted means that the 1˚ fate cell shifts from
P6.p to P5.p or P7.p). These events are probably due to spontaneous failure, or a low level of expression of the let-23 gene
in ksEx10. The incomplete rescue of sy97 or mn23 by the extrachromosomal array, and the incomplete penetrance of sy97
form the background of the results in the mosaic analyses
described below.
let-23 is required for vulval induction in the ABp
lineage but not in the other lineages
The lineages where ksEx10 was lost, and the phenotypes of the
mosaic animals are summarized in Figs 2 and 3. In the sy97
background (Fig. 2), a single ABp mosaic obtained was
vulvaless, and about half of the mosaics in the lineages from
ABp to VPCs showed an abnormal vulval development. In
contrast, the ABa, P1, P2, EMS and MS mosaics were all
normal. There were several mosaic animals without a vulva or
with an abnormal vulva, which had lost ksEx10 in ABar,
ABalpa, E or C. However, these are thought to be within the
spontaneous failures described in the last section. In the mn23
background (Fig. 3), similar results were obtained although no
ABp mosaics and only one ABa mosaic appeared, which is
probably due to the focus of lethality in these lineages, as will
be described later. Thus, let-23 function is required for vulval
induction in ABp but not in other lineages.
let-23 is required cell-autonomously for 1˚ vulval
fate induction but not in 2˚fate VPCs
The six VPCs develop in ABp lineage as shown in Fig. 4.
Therefore, mosaicism in the ABp lineage produces characteristic mosaic patterns in the ventral row of VPCs. Ncl phenotypes
and fates of the VPCs in the mosaics for the ABp lineage are
shown in Tables 2 and 3. When all the VPCs were Ncl, namely
when ksEx10 was lost in all the VPCs, neither 1˚ nor 2˚ fates
were induced (no. 1 in Table 2 or 3). When P6.p was non-Ncl,
2660 M. Koga and Y. Ohshima
Fig. 3. Mosaic analysis of animals carrying a chromosomal
let-23(mn23) lethal mutation, showing foci of the lethality in
ABal and ABpl lineages. See the legend of Fig. 2 for
explanations.
namely when ksEx10 was kept in P6.p, P6.p was induced to the
1˚ fate, as in wild-type animals, in almost all cases (Table 2, no.
28-53 and Table 3, no. 44-93), and not induced to the 1˚ fate in
a few cases (Table 2, no. 54, 55 and Table 3, no. 94, probably
due to a failure in the expression of let-23 on the Ex10).
However, when P6.p was Ncl, P6.p was never induced to the
1˚ fate (Table 2, no. 3-27 and Table 3, no. 2-43). Furthermore,
in these animals, non-Ncl P5.p, P7.p or both, instead of P6.p,
were induced to the 1˚ fate, to form a shifted vulva in 48% and
69% of the cases in the sy97 and mn23 strains, respectively
(Table 2, no. 3-14, Table 3, no. 3-31, and summarized in Table
4). In some animals with Ncl P6.p and non-Ncl P5.p or P7.p,
neither P5.p nor P7.p was induced to the 1˚ fate (e.g. Table 2,
no. 17-27). In these cases, AC signal was probably not strong
enough around P5.p or P7.p. These results show that let-23 is
required cell-autonomously for 1˚ fate induction.
Fig. 4. Detailed lineages for the formation of six VPCs. a, p, l and r
indicate anterior, posterior, left and right daughters, respectively.
P3/4L and P3/4R are produced symmetrically in the left and the right
sides of the animal, respectively. During L1 stage, they move into
the ventral side of the body and lie in a line along the anteriorposterior axis. Both have a 50% probability to be anterior. The
anterior one becomes P3 and the posterior one becomes P4. In the
same manner, P5/6L and P5/6R become P5 and P6, and P7/8L and
P7/8R become P7 and P8. The posterior daughters of P3-8 cells,
P3.p-P8.p, maintain the same order as their mothers in the ventral
row.
Mosaic analysis of let-23 gene function 2661
Table 2. Cell fates of VPCs in VPC mosaics in the
let-23(sy97) vulvaless background
#
*
P3.p
P4.p
P5.p
P6.p
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
j
j
s
s
n
n
n
s
n
n
s
n
e
n
m
m
j
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
p
p
3
2
p
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
3
3
3
(1)
3
p
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
p
3
p
3
P7.p P8.p
3
3
3
3
3
3
3
3
3
1
1
1
1
1
3
3
3
3
3
3
3
3
3
3
3
3
p
2
3
p
3
3
3
3
18
j
3
3
3
3
3
3
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
j
j
j
j
j
j
j
j
j
d
d
d
d
d
d
d
d
m
m
d
d
d
d
d
d
d
d
d
d
d
m
m
d
d
d
j
j
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
p
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
p
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
p
2
2
2
2
2
2
2
2
2
2
2
2
3
3
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
The cell where
Ex10[let-23, ncl-1]
was lost
ABp
ABprappa & P5/6 L
ABprap-ABpra
ABplappa
ABpl
ABplapp
ABprappa
ABprap-ABpra
ABprap-ABpra
ABpr
P6
P6.p
ABpr
P6
ABpr
P6
(P6) : descendants of
P6.a were Ncl
(P6) : descendants of
P6.a were Ncl
ABplap-ABpla
ABprap-ABpra
ABplapp
ABp(l/r)appa
ABprap-ABpra & E
ABplapp-ABpla
ABprapp
ABplapp
ABprap-ABpra
ABpr
ABpr
ABprapp
ABprap-ABpra
ABpr
ABpr & ABa
ABprapp
ABprapp
ABprap-ABpra
ABplappa
ABplapp-ABpla
ABprap-ABpra
ABprapp
ABpr
ABpr
ABplap-ABpla
ABprap-ABpra
ABplap-ABpla
ABprap-ABpra
ABplapp-ABpla
ABprap-ABpra
ABplapp
P5.p
ABprappap
P7 or its mother
P7
ABprap-ABpra
ABprap-ABpra
*Symbols correspond to those of Fig. 2 and Fig. 3 showing vulval
phenotypes: solid circle, normal vulva; solid square, vulvaless; solid triangle,
incomplete vulva; open circle, shifted complete vulva; open triangle, shifted
incomplete vulva; open diamond, shifted multivulva. 1, 2, 3 and p indicate 1°,
2°, 3° and partially induced fates, respectively. Bold and underline typefaces
show non-Ncl and Ncl, respectively. Parentheses mean ambiguity.
In contrast to the 1˚ fate, 2˚ fate cells were produced next to
a 1˚ fate cell regardless of the Ncl phenotypes. When P6.p had
a 1˚ fate, Ncl P5.p and P7.p were induced to 2˚ fate in almost
all cases (Table 2, no. 28-53 except 36, 37, 49, 50 and Table
3, no. 44-92, see also Table 5). When P5.p or P7.p was 1˚, 92%
and 83% of Ncl P6.p were induced to the 2˚ fate in the sy97
and mn23 strains, respectively (Tables 5A and 5B). About 50%
of P4.p and P8.p were also induced to the 2˚ fate regardless of
their Ncl phenotypes in the mn23 strain (Table 5B). Although
the sample number was too small to say exactly, a similar
tendency could be seen in the sy97 strain (Table 5A). Overall,
no 2˚ fate cells, which were not adjacent to a 1˚ fate cell, were
observed in any mosaic animal. These results show that let-23
is not required in 2˚ fate VPCs, and suggest that the 2˚ fate is
induced by a signal from a 1˚ fate cell. These results also
suggest that the graded AC signal is not sufficient for 2˚ fate
induction. The possibility that sy97 or mn23 Let-23 mutant
proteins have enough residual activity to induce the 2˚ fate is
not likely for two reasons. First, let-23(sy97) is a strong hypomorphic allele and let-23(mn23) is probably null (Aroian and
Sternberg, 1991; Herman, 1978; also see All Ncl column in
Table 1). Second, the let-23(mn23) defect is more severe than
that of let-23(sy97), therefore, if the hypothesis were true, 2˚
fate induction should have been more severely affected in
mosaics with let-23(mn23) mutation than in those with let23(sy97), which was not the case (compare Table 5A with 5B).
Foci of the lethality of a let-23 mutation
Since a homozygote of a let-23 null allele dies at the mid-late
L1 stage, let-23 is essential for larval survival as well as for
vulval induction (Herman, 1978; Aroian and Sternberg, 1991).
The cellular basis for the let-23 lethality has remained
unknown. In the let-23(mn23) null background (Fig. 3), there
are two lineages, AB-ABa and AB-ABp-ABpl, in which
somatic loss of ksEx10 was extremely rare. These results mean
that mosaics in these lineages were dead before scoring at
middle L3 to L4. Even for ABa and ABpl lineages, many
mosaics had lost ksEx10 in ABar, ABalpa or in the lineages
from ABpla to VPCs. Thus, some cells in the ABal (except
ABalpa) and ABplp lineages require let-23 gene function for
survival. In the let-23(sy97) reduced function background (Fig.
2), mosaics in the AB-ABp-ABpl lineage were as rare as in the
mn23 background, whereas many ABa mosaics were scored.
Therefore, the focus of lethality of let-23(sy97) exists in ABplp
but not in ABal, although about 90% of let-23(sy97) larvae are
lethal and the phenotype of the dead larvae looks the same as
for let-23(mn23). The difference between the foci of lethality
of sy97 and mn23 may be due to a difference in requirements
of amount or quality of LET-23 between ABal and ABplp.
DISCUSSION
We obtained three major findings in our mosaic analyses. First,
let-23 is required cell-autonomously for induction of the 1˚
vulval fate. Second, let-23 is not required cell-autonomously
in a 2˚ fate VPC. Third, the 2˚ fate is induced in VPCs adjacent
to a 1˚ fate cell, regardless of the presence or absence of the
let-23 wild-type gene. The first finding supports the idea that
LET-23 is a receptor for the vulval induction signal from the
AC, and shows that the so-called vulval induction pathway
including LET-23 as a pivotal component (which is referred to
as the let-23 pathway below) works to induce the 1˚ vulval fate
2662 M. Koga and Y. Ohshima
Table 3. Cell fates of VPCs in VPC mosaics in the
let-23(mn23) lethal background
#
*
P3.p
P4.p
P5.p
P6.p
P7.p P8.p
1
j
3
3
3
3
3
3
2
j
3
3
3
3
3
3
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
e
e
e
e
e
e
e
e
s
s
s
s
s
s
s
s
s
n
n
n
n
n
n
s
s
s
n
n
n
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
p
3
3
p
p
3
2
2
2
2
2
2
2
2
2
p
p
p
p
p
3
3
3
3
3
3
3
1
1
1
1
1
1
1
p
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
2
2
2
2
p
2
p
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
p
2
2
2
2
2
3
1
1
1
1
1
p
p
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
3
3
2
p
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
p
p
3
32
33
34
35
n
j
j
j
3
3
3
3
3
3
3
3
3
3
3
3
p
3
3
3
p
3
3
3
3
3
3
3
36
37
j
j
3
3
3
3
3
3
3
3
3
3
3
3
38
39
40
41
42
j
j
j
j
j
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
43
j
3
3
3
3
3
3
44
45
46
47
48
49
50
51
52
53
54
55
56
57
d
d
d
d
d
d
d
d
d
d
d
d
d
d
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
The cell where
Ex10[let-23, ncl-1]
was lost
ABpr & ABpla &
ABar
ABplap-ABpla &
P5 or P6
ABplap-ABpla
ABpl & ABa & EMS
ABplapp
P6.p & E
P6.p
P6
ABprap-ABpra
ABpr
ABpla
ABpla
ABplapp
ABprapp
ABplapp
ABprap-ABpra
ABpr
ABplap-ABpla
ABprap-ABpra
ABprapp
ABplap-ABpla
ABp(l or r)appaa
ABplap-ABpla
ABplapp
ABprap-ABpra
ABprap-ABpra
ABprapp
P6
P6.p
P6
(P6) : descendants of
P6.a were Ncl
P6
ABplapp
ABp(l or r)appa
ABprap-ABpra &
MS
ABplapp
(P6) & (P7 or its
mother) :
descendants of
P6.a and P7.a were
Ncl
ABprapp
ABplapp
ABp(l or r)appaa
ABplapp
ABplappap & P6 :
descendants of
P6.a were Ncl
(P6) : descendants of
P6.a were Ncl
ABprap-ABpra
ABpr
ABprap-ABpra
ABpr
ABprap-ABpra
ABp(l or r)appa
ABpr & P1
ABprap-ABpra
ABprapp
ABprap-ABpra
ABpr
ABpr
ABprap-ABpra
ABpr
#
*
P3.p
P4.p
P5.p
P6.p
P7.p P8.p
58
59
60
61
62
63
64
65
66
d
d
d
d
d
d
d
d
d
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
m
s
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
p
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
The cell where
Ex10[let-23, ncl-1]
was lost
ABplap-ABpla
ABprapp
ABplapp
ABplapp
ABprapp
ABprapp
ABplap-ABpla
ABpr
ABplap-ABpla &
ABalpa
ABprapp
ABplapp
ABplap-ABpla
ABprap-ABpra
ABplap-ABpla
ABplapp & ABalpa
ABplap-ABpla
ABpr
ABprap-ABpra
ABprap-ABpra
P5.p
P5
P5
P5
P5.p
P5
P7.p
P7.p
P7 or its mother
P7 or its mother
P7.p
P7
P7 or its mother
P7
P7
P7
ABpr
P4.p
See the legend for Table 2.
Table 4. 1° fate induction in mosaic animals with Ncl P6.p
and non-Ncl P5.p or P7.p
Number and (%) of animals
1° fate induction
let-23(sy97)
background
let-23(mn23)
background
In both of P5.p and P7.p
In either of P5.p or P7.p
Partially in either of P5.p or P7.p
In none of the VPCs
Others
Total
1
11
1
12
0
25
5
24
1
12
0
42
4
44
4
48
0
100
12
57
2
29
0
100
in a cell-autonomous manner. Despite the second finding, it is
clear that the let-23 pathway is required for both 1˚ and 2˚ fate
induction since reduced function mutations of let-23, as well
as other genes from lin-3 to mpk-1 in let-23 pathway, abolish
both 1˚ and 2˚ fate induction. The third finding indicates that
a signal from a 1˚ fate cell induces its neighbors to adopt the
2˚ fate, independently of let-23.
The model, which was proposed by Sternberg and Horvitz
(1989) based on genetic studies on the interaction among
mutations of lin-12, Vul genes (lin-3, lin-2, lin-7 and lin-10)
and a Muv gene (lin-15), could basically explain the results
Mosaic analysis of let-23 gene function 2663
A
B
Fig. 5. (A) Induction of vulval cell fates in a wild-type hermaphrodite. The six equivalent VPCs (P3.p-P8.p) are located ventral to the gonad.
AC is just above P6.p in the gonad. The lineage of cells derived from each VPC is shown at the bottom (Sulston and Horvitz, 1977). (B) A
model for intercellular signalings in vulval induction. Arrows indicate activation and a T-bar indicates inactivation. The thick arrows and T-bar
mean stronger signals, and thin arrows weaker signals. The dotted arrows represent signals which are very weak or absent usually, because of
down regulation by the lateral signal from the 1˚ fate VPC.
above. Their model includes a graded inductive signal from the
AC and a lateral signal among VPCs as basic components. Fig.
5B shows our model designed to explain the mosaic results
reported here. In this model, two points are revised: the
autocrine element of the lateral signaling pathway is removed
(this is discussed just below), and the branch point of a signal
to activate the lateral signal is placed in the later part (after lin1) of the let-23 pathway (this may be minor, mentioned in the
next paragraph). The original model considers autocrine in
addition to paracrine modes for the action of the lateral signal
to induce the 2˚ fate. Autocrine signaling was assumed mainly
because an isolated, slightly AC-distal VPC adopted the 2˚ fate
and such a VPC was considered to receive the vulval induction
signal of an intermediate level (Sternberg and Horvitz, 1986).
In contrast to their results, we did not find any independently
induced 2˚ fate cell in our mosaic experiments. The critical difference between these two experiments may be the presence of
cell-cell contact between the VPCs in the mosaic experiments
and its absence in the isolated VPC experiments. Based on this
difference, it is possible to explain the above difference in 2˚
fate induction. For example, in the presence of cell-cell
contact, which is a normal situation, autocrine lateral signaling
2664 M. Koga and Y. Ohshima
Table 5A. Fate of the VPCs neighbouring a 1° fate VPC in
mosaic animals with let-23(sy97) mutation
P4.p
P5.p
nonNcl Ncl
nonNcl Ncl
P6.p
P7.p
P8.p
Ncl
nonNcl
1°
2°
p°
3°
0
2
1
0
0
1
2
1
0
3
0
0
0
21
1
1
0
0
0
0
0
11
1
0
0
11
0
2
0
12
1
0
0
1
0
2
0
0
2
0
Total
3
4
3
23
0
12
13
13
3
2
Fate
nonNcl
Ncl
nonNcl Ncl
Numbers represent those of animals.
Table 5B. Fate of the VPCs neighbouring a 1° fate VPC in
mosaic animals with let-23(mn23) mutation
Fate
1°
2°
p°
3°
Total
P4.p
P5.p
nonNcl Ncl
nonNcl Ncl
P6.p
nonNcl
P7.p
P8.p
Ncl
nonNcl
0
6
4
3
0
5
4
0
0
10
0
0
0
39
1
0
0
1
0
0
0
25
4
1
0
22
0
1
0
27
0
0
0
1
3
2
0
2
0
2
13
9
10
40
1
30
23
27
6
4
Ncl
nonNcl Ncl
does not function effectively, yet in the absence of cell contact,
it does function. The mechanism regulating the autocrine
pathway is unclear and other explanations could be possible.
We believe that the mosaic results are more likely to reflect the
events occurring in the intact animal than those obtained with
isolated VPCs, because let-23 mosaic animals have the same
number and position of VPCs as those of wild-type animals
until the fates of the VPCs are determined.
According to our revised model, vulval induction processes
in wild-type animals are as follows (Fig. 5B). Vulval induction
signal secreted from the AC (AC signal) forms a gradient. The
most plausible candidate for the AC signal is LIN-3. Since
LIN-3, as deduced from its cDNA, has a putative transmembrane domain, posttranslational processing may be needed to
release LIN-3 from the cell membrane (Hill and Sternberg,
1992), although no biochemical evidence for processing of
LIN-3 was obtained. In the gradient of the AC signal, the concentration is high enough around P6.p to induce the 1˚ fate,
intermediate around P5.p and P7.p, and too low around P3.p,
P4.p and P8.p. This idea is supported by the results that nonNcl P6.p were induced to the 1˚ fate in about 100% of the VPC
mosaic animals, whereas non-Ncl P5.p or P7.p were induced
to the 1˚ fate in 48% and 69% of the mosaics, along with Ncl
P6.p, in the sy97 and mn23 strains respectively, and that P3.p,
P4.p and P8.p were not induced in any mosaic (Tables 2, 3 and
4). In P6.p, LET-23 is activated by binding of the AC signal
molecule causing it to adopt the 1˚ fate probably through the
let-60Ras-mpk-1MAPK signal transduction pathway. For 1˚
fate induction in P6.p, LIN-3 membrane bound form on the AC
might function (Hill and Sternberg, 1992) since P6.p is closest
to the AC. LET-23 activation also triggers the lateral signal to
induce the 2˚ fate in adjacent cells. In P5.p and P7.p, although
LET-23 could also be activated by an intermediate AC signal,
the lateral signal from the P6.p, which has been activated more
strongly or earlier by the AC signal, counteracts 1˚ fate
induction either through induction of the 2˚ fate or directly.
The main pathway described above is shown by thick arrows
in Fig. 5B. At an early phase of induction, there may be a
period when P5.p - P7.p cells compete with each other to
induce their neighbors to the 2˚ fate through the lateral signal
(shown by dotted lines in Fig. 5B). Even if this is the case, such
a period would be short, otherwise P4.p or P8.p might be
induced to the 2˚ fate by the lateral signal from P5.p or P7.p.
When P6.p is let-23−, the usual main pathway does not work,
and instead the minor pathways (shown by thin or dotted lines
in Fig. 5B), which are usually hidden or repressed behind the
main pathway, become predominant. Therefore, in mosaic
animals with Ncl P6.p, P5.p and P7.p could adopt the 1˚ fate
and induce adjacent VPCs to the 2˚ fate.
The lateral signaling pathway may branch from the pathway
inducing 1˚ fate after lin-1, because a mutation of lin-1, which
encodes a putative transcription factor and is placed far downstream of the vulval induction pathway, induces an additional
1˚ fate cell together with a 2˚ fate cell in P3.p and P4.p
(Ferguson et al., 1987). As to the lateral signaling pathway, the
molecular basis is unknown except for lin-12, which has been
proposed to be a receptor for the lateral signal (Sternberg and
Horvitz, 1989). Lag-2 was identified as a ligand of Lin-12 in
AC/VU decision (Tax et al., 1994, Henderson et al., 1994).
However, it is unknown whether Lag-2 also acts as a ligand
for Lin-12 in vulval induction. It is also unknown whether a
Ras-MAPK pathway is involved in the lateral signaling or not.
The 2˚ fate is induced in VPCs adjacent to a 1˚ fate cell.
However, the frequency of 2˚ fate induction varies among
VPCs. The frequency is highest in P5.p and P7.p (nearly
100% and about 90% in the mn23 and sy97 strains, respectively), less in P6.p (84 and 92%) and low in P4.p and P8.p
(50-30%) (Tables 5A and B). This M shaped spectrum could
be explained as follows. The amount of lateral signal given
by a 1˚ fate cell could correlate with the amount of the AC
signal received. Since P6.p, the VPC nearest to AC, can be
activated most strongly by the AC signal, the lateral signal
released from P6.p would be the strongest when P6.p adopts
the 1˚ fate. Therefore, P5.p and P7.p are induced to the 2˚ fate
most strongly. In contrast, since P5.p or P7.p cannot be
activated as strongly as P6.p by the AC signal, the lateral
signal from them may be weaker than that from P6.p, even if
they can adopt the 1˚ fate. Therefore, P6.p, P4.p and P8.p are
induced to the 2˚ fate at a lower frequency than are P5.p and
P7.p. The probability for 2˚ fate induction is much higher in
P6.p than in P4.p and P8.p perhaps because P6.p can receive
the lateral signal from both P5.p and P7.p while P4.p or P8.p
can receive the signal from only P5.p or P7.p. However, since
induction of both P5.p and P7.p to the 1˚ fate was not frequent
in mosaics with a Ncl P6.p (shifted multivulva or diamonds
in Tables 2 and 3), a signal that facilitates a VPC to take the
2˚ fate in response to the lateral signal might be secreted from
the AC.
In the course of our mosaic analysis, the foci of let-23(mn23)
lethality were found in the ABal (except ABalpa) and ABplp
lineages. This means that let-23 may play a critical role in the
fate or survival of neurons since the cells that derive from ABal
(except those from ABalpa) are all neurons. In hermaphrodites,
only three neurons, CANL, CANR and M4 are known to be
essential for survival (Chalfie and White, 1988), and CANL
and CANR derive from the ABala lineage. Therefore, defects
in CANL and CANR neurons may be responsible for the
Mosaic analysis of let-23 gene function 2665
lethality of the let-23 mutation. Although the function of CAN
neurons is unclear, it is known that their axons run along with
the excretory cell, which is a big H shaped cell thought to
control osmotic pressure (Nelson et al., 1983; Nelson and
Riddle, 1984; White, 1988). Interestingly, the excretory cell is
born in the ABplp lineage. Therefore, the lethality of let-23
mutations may be due to a defect in the control of osmotic
pressure. In dead larvae of let-23 mutants, internal tissues look
shrunken and detached from the body wall, perhaps due to
abnormal osmotic pressure. However, there is no direct
evidence to show which cell requires let-23 activity for
survival. Identification of the cells expressing let-23 is needed
to examine these possibilities.
Our let-23 mosaic experiments revealed a sequential
induction mechanism, where the let-23 pathway first induces
the 1˚ fate and then the lateral signaling pathway induces the
2˚ fate. The lateral signaling pathway requires further study to
better understand molecular mechanisms involved in vulval
induction.
We thank D. Waring for the ncl-1 cosmid clone, R. Aroian, J.
Mendel and P. Sternberg for let-23 mutant strains and communicating unpublished information, C. Kari and R. Herman for the ncl-1
mutant strain and M. Ohara for language assistance. Some nematode
strains used in this work were provided by the Caenorhabditis
Genetics Center, which is funded by the NIH National Center for
Research Resources (NCRR). We especially thank S. Kim for unpublished results and suggestions about let-23 mosaic analysis. We are
grateful to I. Mori, K. Ogura, H. Honda and other members of our
laboratory for nematode strains and stimulating discussions. This
research was supported by a grant from the Ministry of Education,
Science and Culture of Japan and a grant from the Science and Technology Agency of Japan.
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(Accepted 1 May 1995)