Download HOM-C/Hox genes and four interacting loci

Development 120, 2579-2593 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
2579
HOM-C/Hox genes and four interacting loci determine the morphogenetic
properties of single cells in the nematode male tail
King L. Chow* and Scott W. Emmons†
Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
*Present address: Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
†Author for correspondence
SUMMARY
The copulatory structure of the C. elegans male tail
includes a set of nine bilaterally symmetrical pairs of sense
organs known as rays. Each ray comprises three cells,
which are generated by a stereotyped cell sublineage
expressed by 18 epidermal ray precursor cells. A pattern
formation mechanism in the epidermis guides the specification of morphogenetic differences between the rays
necessary for correct organelle assembly at specific
positions within the epidermis. Expression of these ray differences was altered in mutations we described previously,
resulting in displaced and fused rays. Here we show that
two genes of the C. elegans HOM-C/Hox gene complex play
a role in the pattern formation mechanism. Increasing or
decreasing the gene dosage of mab-5, an Antennapedia
homolog, and egl-5, an Abdominal B homolog, results in
displacement and fusion of specific rays. These changes are
interpreted as anterior or posterior transformations in ray
identities. Mutations in the genes previously described are
dominant modifiers of these effects. This suggests that these
genes act in the same morphogenetic pathway as mab-5 and
egl-5. Several lines of evidence, including cell ablation
experiments, argue that the identity of each ray is specified
cell-autonomously in the terminal cells of the ray lineages.
mab-5 and egl-5, therefore, specify the morphogenetic
properties of differentiating cells, without change in cell
lineage or apparent cell type. Modifier genes may act
upstream of mab-5 and egl-5 to regulate their expression.
Alternatively, they may act at the same step in the pathway,
as cofactors, or they may be target genes. Target genes
could include genes specifying cell recognition and
adhesion molecules governing ray organelle assembly.
INTRODUCTION
for organelle assembly at reproducible positions. Here we
show that two genes encoding Antennapedia-class homeodomain transcription factors, members of the C. elegans
HOM-C/Hox gene cluster, as well as several genetic modifiers
of HOM-C/Hox gene mutations, play a role in the pattern
formation mechanism.
The C. elegans epidermis consists of a large syncytium
covering most of the body, distinctly shaped cells and syncytia
constructing the head and tail regions, and left and right lateral
rows of seam cells (White, 1988). The male-specific sense
organs that are the subject of this paper are generated by seam
cells. At hatching, there are nine seam cells arrayed in single
file on each side of the body. During postembryonic development, seam cells generally divide in a stem cell pattern to
generate an anterior daughter that fuses with the large hypodermal syncytium, and a posterior daughter that remains as a
seam cell (Sulston and Horvitz, 1977; Fig. 1A). In certain
regions of the body, however, seam cells generate neuroblasts.
Seam cell lineages can therefore serve as a model to study the
mechanism of axial pattern formation in C. elegans. During the
L1 larval stage in both sexes, the embryonic seam cell T
generates the phasmid, a chemosensory sensillum. During the
L2 larval stage in both sexes, a descendant of the embryonic
seam cell V5 generates the postdeirid. The postdeirid is a
Two central processes in the development of metazoans are the
production of multiple cell types and the morphogenesis of
complex multicellular structures. Morphogenesis requires that
reproducible homotypic and heterotypic cellular contacts and
interactions be established following precise cell recognition
events. In such morphogenetic interactions, cells of a single
overt cell type may not all act the same. Pattern forming mechanisms that generate non-equivalent cell properties thus play a
crucial role in establishment of the form of the organism. This
process is particularly acute in development of the nervous
system, where neurons acquire distinct characteristics
necessary to generate a complex network of cell contacts.
We have been studying development and morphogenesis of
the copulatory structures of the C. elegans male tail (Emmons,
1992). Because its cellular structure is completely defined
(Wood, 1988), C. elegans provides an opportunity for investigating, in detail, the relationship between pattern formation and
morphogenesis. We have shown previously that the distinctive
arrangement of sense organs in the male tail is dependent on a
pattern formation mechanism in the epidermis (Baird et al.,
1991). This pattern formation mechanism generates differences between cells of the peripheral nervous system necessary
Key words: C. elegans, morphogenesis, homeobox, HOM-C/Hox
genes, neurogenesis
2580 K. L. Chow and S. W. Emmons
E
Ray
1
2
3
4
5
6
7
8
9
Shape
Position
of opening
Dopamine
Dilated
cisternae
in neuron B
thin
thin
thin
thin
thin
thick, tapered
thin
thin
thin
dorsal
ventral
marginal
ventral
dorsal
absent
dorsal
ventral
marginal
−
−
−
−
+
−
+
−
+
+
+
+
+
+
−
+
+
+
Fig. 1. Development of nine distinct
sensory rays. (A) Postembryonic seam
cell lineages leading to the ray
precursor cells, Rn (n=1-9) (Sulston
and Horvitz, 1977). V5, V6 and T
denote the three most posterior seam
cells generated during embryogenesis.
All the cells of the lineages shown lie
in the seam, a single-file lateral row of
cells extending from the head to the tail
on each side of the animal, with the
following exceptions: cells marked h
fuse with the hypodermal syncytium;
cells marked PDE generate the
postdeirid, a neuronal structure of
unknown function; cells marked PH
contribute to the phasmid, a posterior
sensory structure. Cells marked X
undergo programmed cell death. The
scale to the left indicates hours of
postembryonic development after
hatching, as well as the various larval
stages through which the animal
passes. (B) Ray precursor cells R1-R9
in an L3 male; the cell nuclei with
visible nucleoli are indicated. The
positions of the V6.a nucleus (arrow)
and of the anus (open arrowhead) are
indicated for orientation. Nomarski
photomicrograph. Scale bar indicates
10 µm. (C) The ray sublineages
generated by the ray precursor cells.
The sublineage generates five cell
types as follows: Rn.aaa: A-type
neuron; Rn.aap: programmed cell
death; Rn.apa: B-type neuron; Rn.app:
support cell; Rn.p: hypodermal cell
(Sulston et al., 1980). The Rn.p
hypodermal cells fuse together to form
the tail seam (SET), or fuse with the
hypodermal syncytium. (D) Adult male
tail, ventral view, showing the nine
pairs of rays embedded in the acellular
fan. Nomarski photomicrograph. Scale
bar indicates 10 µm. (E) Distinguishing
properties of the rays. Shape refers to
the overall profile of the ray as it
appears in the adult; position of
opening refers to the side of the fan on
which the ray opens (ray 6 is not open
to the exterior); dopamine refers to
whether or not the A-type neuron
expresses dopamine (Sulston et al.,
1980); B neuron cisternae refers to the
presence or absence of dilated cisternae
within the cell body of the B-type
neuron (Sulston et al., 1980).
Role of HOM-C/Hox genes in morphogenesis 2581
neuronal structure of unknown function. During the L3 larval
stage in males, descendants of V5, V6, and T generate ray neuroblasts, termed ray precursor cells (Rn cells) (Fig. 1B). Each
ray precursor cell expresses the ray sublineage, and gives rise
to the cells of one ray (Fig. 1C). The rays are male-specific
sensilla that extend outward from the tail in an acellular fan
(Fig. 1D). They are necessary for copulation. As the rays differ
from each other, there is further axial patterning within the tail
region (Fig. 1E).
Each ray comprises the dendritic endings of two neurons and
a support cell. The ends of the rays are located at reproducible
positions in the cuticle, giving the male a characteristic,
species-specific posterior morphology (Fig. 1D). In our
previous studies we described the cellular mechanisms
involved in the development of the adult ray pattern, and
demonstrated a role of six genes essential to the process (Baird
et al., 1991). From these studies we concluded that the rays
were not morphogenetically equivalent. In spite of the fact that
they arose from repetition of a stereotyped cell sublineage
(Sulston and Horvitz, 1977) (Fig. 1C) and comprised cells of
the same three cell types (Sulston et al., 1980), each ray
behaved differently in its interactions with surrounding
epidermal cells. Furthermore, cells of individual rays were able
to recognize each other during organelle assembly, suggesting
that they expressed distinct cell recognition functions. Both
these properties were lost in mutants, resulting in misplacement of rays and fusion of one ray to another. The rays also
differ in their individual morphology, their ultrastructure, and
their neurotransmitter usage (Fig. 1E) (Sulston and Horvitz,
1977; Sulston et al., 1980). Thus in development of the rays
we see an example of the specification of differences among
cells of a single differentiated cell type, and of the dependence
of morphology on these differences.
We report that mutations in two genes of the C. elegans
HOM-C/Hox gene cluster, mab-5 and egl-5, affect the morphology of particular rays. In C. elegans, homeotic selector
genes are organized into a gene cluster similar to the HOMC/Hox clusters of fruitflies and vertebrates (Clark et al., 1993;
Wang et al., 1993; McGinnis and Krumlauf, 1992). mab-5 is
most similar to Drosophila HOM-C genes Antennapedia,
Untrabithorax, and AbdominalA, while egl-5 is most similar
to Drosophila HOM-C gene AbdominalB. In addition to mab5 and egl-5, the C. elegans HOM-C gene cluster contains the
gene lin-39, which shares similarities to Drosophila proboscipedia, Sex combs reduced, and Deformed, and ceh-13, which
shares similarities to Drosophila labial. Mutations in the C.
elegans genes, with the exception of ceh-13, have been shown
to affect the division patterns, fates, migration, fusion, and neurotransmitter expression of cells in particular body regions.
We describe a role of mab-5 and egl-5 in determining additional morphogenetic properties of cells. In particular, we show
that raising and lowering the gene dosage of mab-5 and egl-5
results in changes in ray properties that appear to reflect
specific transformations of one ray morphogenetic type into
another. For two of the six most anterior rays, ray morphology
is influenced by the ratio of the number of copies of these two
genes. Action of mab-5 and egl-5 in the different terminal
branches of the ray lineages appears to be cell-autonomous, but
the characteristic level of expression of these genes postulated
to define each lineage branch may be influenced by interactions between seam cells. Mutations in several of the genes
described previously are shown to be dominant modifiers of
mutations in mab-5 and egl-5. This suggests that their action
is related to implementing the pattern formation mechanism.
MATERIALS AND METHODS
Nematodes
All nematodes used in this work were derivatives of strain Bristol,
N2. They were reared following standard methods (Brenner, 1974;
Sulston and Hodgkin, 1988). Unless otherwise noted, animals were
grown at 20°C.
Most bx alleles of mab genes were isolated by EMS mutagenesis
(Baird et al., 1991; this work), following the mutagenesis protocol of
Brenner (1974). bx80 was isolated after mutagenesis with 10−4 M
1,2,3,4-di-epoxy-butane (DEB) (Trent et al., 1991). The strains used
generally carried the him-5(e1490) mutation, which elevates X chromosome non-disjunction (Hodgkin et al., 1979). The purpose was to
increase the frequency of spontaneous males among the progeny of
selfing hermaphrodites.
Strains bearing the following additional mutations were used:
Linkage group I: mab-20(bx24), mab-20(bx61ts)
Linkage group II: tra-2(q279), mnC1[dpy-10(e128)unc-52(e444)]
Linkage group III: egl-5(n486), egl-5(n945), mab-5(e1751), mab5(bx54), mab-5(e1239), mab-21(bx53), mab-21(bx41), mab21(sy155), qDp3, pal-1(e2091), unc-36(e251)
Linkage group IV: dpy-9(e12), lin-22(mu2), mab-26(bx80), unc17(e245)
Linkage group X: mab-18(bx23)
Properties of these mutations have been or are described as follows:
egl-5(n486) and egl-5(n945), Chisholm (1991); lin-22(mu2), Waring
et al. (1992); mab-5(e1239), Kenyon (1986); mab-5(bx54), this work;
mab-5(e1751), Hedgecock et al. (1987); alleles of mab-18 and mab20, Baird et al. (1991); alleles of mab-21, Baird et al. (1991) and Chow
and Emmons (in preparation); mab-26(bx80), this work; pal1(e2091), Waring and Kenyon (1990). For dpy-9(e12), unc-17(e245),
tra-2(q279), unc-36(e251), mnC1, and qDp3 see Hodgkin et al.
(1988). Additional known properties of the mab-5 and egl-5 alleles
relevant to the work described here are presented in Table 1.
Isolation and mapping of mab-5(bx54)III and mab26(bx80)IV
The bx54 and bx80 mutations were isolated by screening for morphological defects among male F2 progeny of him-5(e1490) hermaphrodites mutagenized with EMS (bx54) or DEB (bx80) (Brenner,
1974). The bx54 mutation was identified as a mutation causing loss
of anterior rays. It was mapped to LG III and shown to be an allele
of mab-5 by failure to complement mab-5(e1239). S. Salser and C.
Kenyon have shown that bx54 is a E(135)→K missense mutation in
the homeobox of mab-5 (personal communication).
The semi-dominant bx80 mutation was identified by its ray fusion
phenotype: in homozygotes rays 1 through 6 are variably fused
together. Males and hermaphrodites also have deformities of the body
in the nose, posterior midbody, and tail regions. mab-26(bx80) was
placed near to or to the left of dpy-9 on the left end of LGIV by a
three factor cross with dpy-9 and unc-17 (data not shown).
Dosage analysis of HOM-C/Hox genes and their genetic
modifiers
Animals heterozygous for two unlinked genes were analyzed by
mating him-5(e1490) males to homozygous double mutants and
scoring the ray phenotype of cross progeny males. For the linked
genes mab-21, mab-5, and egl-5, doubly heterozygous strains were
first constructed by mating single mutants together. Lines segregating
both homozygous and double heterozygous progeny were maintained
and heterozygous males were scored for ray phenotype. For strains
2582 K. L. Chow and S. W. Emmons
Table 1. Properties of mab-5 and egl-5 alleles
Allele
Molecular structure
egl-5
n486
Genetic properties
References
R52→C within the homeodomain
slightly weaker phenotype than the
null mutation u202, presumably
a strong hypomorph
Chisholm, 1991; Wang et al., 1993
not known
amber suppressible, phenotype
same as u202, presumed null
Chisholm, 1991
mutation of the splice donor of the
first intron, which results in
out-of-frame splicing
presumed null
S. Salser and C. Kenyon, personal
communication
bx54
E135→K within the homeodomain
hypomorph
S. Salser and C. Kenyon, personal
communication; this work
e1751
tandem duplication of mab-5
hypermorph or neomorph, results in
ectopic expression of MAB-5 protein
and ectopic expression of mab-5dependent cell fates, not suppressed
by a deficiency in trans.
Salser and Kenyon, 1992; Salser et al.,
1993; K. L. Chow, unpublished
observations
n945am
mab-5
e1239
Table 2. Genetic interactions between mutations affecting ray 4 and ray 6
HOM-C/Hox genotype
1
2
3
4
5
6
7
8
9
10
11
12
+egl-5(n486)/+egl-5(n486);qDp3
+egl-5(n486)/mab-5(e1751)+
+egl-5(n486)/mab-5(e1239)egl-5(n945);qDp3
+egl-5(n486)/++
++/++;qDp3
++/++
+egl-5(n486)/mab-5(bx54)+
+egl-5(n486)/mab-5(e1239)+
mab-5(bx54)+/++
mab-5(e1239)+/++
mab-5(e1239)+/mab-5(e1239)egl-5(n945);qDp3
mab-5(e1239)+/mab-5(e1239)+;qDp3
mab-5/
egl-5
Percentage ray transformation
mab genotype
3/>1
>2/>1
2/>1
2/>1
3/3
2/2
>1/>1
1/>1
>1/2
1/2
1/2
1/3
4→3
6→4
0 (0/229)
0 (0/227)
0 (0/243)
0 (0/272)
3.0 (7/228)
0 (0/>200)
1.3 (5/377)
5.7 (20/351)
5.8 (13/223)
11.5 (29/254)
30.5 (87/285)
62.2 (191/307)
34.1 (78/229)
13.2 (30/227)
16.0 (39/243)
7.0 (19/272)
0 (0/228)
0 (0/>200)
0 (0/377)
0 (0/351)
0 (223)
0 (0/254)
0.4 (1/285)
0 (0/307)
Effect of mab-18 gene dosage
13
14
15
+egl-5(n486)/++
++/++
++/++
2/1
2/2
2/2
2.2 (5/224 )
1.6 (4/254)
2.7 (8/294)
29.2 (67/224)
1.6 (4/254)
6.1 (18/294)
1/2
mab-18(bx23)/+
mab-18(bx23)/+
mab-21(bx53)/+;
mab-18(bx23)/+
mab-18(bx23)/+
16
mab-5(e1239)+/++
17
18
19
20
21
22
23
24
25
Effect of mab-21 gene dosage
+egl-5(n486)/mab-5(e1751)+
+egl-5(n486)/++
++/++
++/++
++/++
+egl-5(n486)/mab-5(bx54)+
+egl-5(n486)/mab-5(e1239)+
mab-5(bx54)+/++
mab-5(e1239)+/++
15.3 (38/248)
3.6 (9/248)
>2/>1
2/>1
2/2
2/2
2/2
>1/>1
1/>1
>1/2
1/2
mab-21(bx53)/+
mab-21(bx53)/+
mab-21(bx53)/+
mab-21(bx41)/+
mab-21(sy155)/+
mab-21(bx53)/+
mab-21(bx53)/+
mab-21(bx53)/+
mab-21(bx53)/+
0 (0/221)
0 (0/367)
0 (0/272)
0.6 (1/179)
0 (0/152)
0.9 (3/341)
7.6 (24/316)
12.3 (56/453)
15.3 (34/219)
40.7 (90/221)
31.6 (116/367)
0 (0/272)
0 (0/179)
0.7 (1/152)
3.5 (12/341)
7.6 (24/316)
0 (0/453)
0 (0/219)
+egl-5(n486)/++
++/++
mab-5(e1239)+/++
2/>1
2/2
1/2
mab-20(bx24)/+
mab-20(bx24)/+
mab-20(bx24)/+
0.8 (2/256)
0.4 (1/232)
12.4 (34/273)
0.8 (2/256)
0.4 (1/232)
0 (0/273)
+egl-5(n486)/++
++/++
mab-5(e1239)+/++
2/>1
2/2
1/2
mab-26(bx80)/+
mab-26(bx80)/+
mab-26(bx80)/+
5.2 (13/251)
24.7 (43/174)
73.9 (190/257)
Effect of mab-20 gene dosage
26
27
28
Effect of mab-26 gene dosage
29
30
31
0 (0/251)
0 (0/274)
0 (0/257)
The symbols >1 are used to represent the gene dosage in genotypes containing egl-5(n486) and mab-5(bx54) to reflect the fact that these mutations are
hypomorphs, and as such provide some gene activity (see Table 1). It is also used to indicate that the mab-5(e1751) mutation appears to increase the level of
mab-5 activity.
Role of HOM-C/Hox genes in morphogenesis 2583
containing the duplication qDp3, either the chromosomal mutation
unc-36(e251), which is covered by qDp3, was used to score for the
presence of the duplication (Table 2, lines 1 and 5), or the presence
of qDp3 was indicated by the presence of a full complement of rays
and normal gross tail morphology (Table 2, lines 3, 11, and 12). To
score the effect of heterozygosity of the X-linked gene mab-18, the
transformer mutation tra-2(q279) II was used. XX animals, normally
hermaphrodites, are transformed into phenotypic males by the
recessive tra-2(q279) mutation. tra-2(q279) was maintained in heterozygous strains balanced by the inversion chromosome mnC1; such
strains segregated one quarter tra-2 homozygous males. tra-2 males
were mated to tra-2/mnC1; mab-18 hermaphrodites, and crossprogeny males, identified as males with at least one wild-type ray 6,
were scored for ray fusion phenotype.
Microscopy, cell lineage analysis and laser ablation
Morphology of adult males and cell lineages were determined by
Nomarski microscopy with a Zeiss Axioskop or Axioplan microscope. For scoring ray fusion, males were mounted by the Standard
Mount procedure (Sulston and Hodgkin, 1988) without bacteria and
observed at 400×. For cell lineage analysis, the coverslip was spread
with bacteria, and animals were observed at 1000×. For laser ablation,
the Anesthetic Mount was used (Sulston and Hodgkin, 1988). Cells
were killed by multiple pulses with a VSL337 nitrogen laser equipped
with a dye laser module containing 440 nm coumerin dye (Laser
Science Inc., Cambridge, MA; see Avery and Horvitz, 1987). The
laser beam was directed into the incident-light port of the Axioplan
microscope with a VSL-LMA Laser Microscope Adapter (Laser
Science Inc., Cambridge, MA).
RESULTS
Decreased mab-5 function results in transformation
of the morphology of ray 4 to that of ray 3
The C. elegans HOM-C/Hox gene mab-5 is required for generation of rays by descendant cells of seam cells V5 (ray 1)
and V6 (rays 2-6), but not T (rays 7-9) (Kenyon, 1986) (Fig.
1). We found that the level of expression of mab-5 also affects
the morphology of some of the rays descended from V5 and
V6. (The rays generated by V5 and V6 will be referred to collectively as V-rays.) In males homozygous for strong mab-5
loss-of-function alleles, the ray sublineage is not expressed in
the V5 and V6 lineages, and the L4 larval seam cells instead
generate adult cuticular structures known as alae, as do
lineages generated by more anterior seam cells (Kenyon,
1986). As no V-rays are generated, the role of mab-5 in specifying ray morphology could not be assessed in this background. In our screens for mutations affecting male tail morphology (Materials and Methods), we identified a weak allele
of mab-5 that allowed us to overcome this difficulty.
The mab-5 mutation bx54 is a hypomorph by the criteria that
it causes loss of only some V-rays, and affects ray morphology
in a manner similar to a mab-5(0)/mab-5(+) heterozygote
(described below). In mab-5(bx54) homozygotes, only anterior
V-rays are lost, and it is possible to examine the role of mab-5
in specification of the morphologies of the remaining rays. By
morphological criteria, mab-5(bx54) homozygous males lack
rays 1, 2, and 4 (Fig. 2). However, by cell lineage analysis we
determined that V5.ppppp, V6.papap, and V6.pappp, the normal
precursors of rays 1, 2, and 3 (Fig. 1), failed to express the ray
sublineage in mutant worms (6/6 sides examined). The most
anterior ray was generated by V6.pppap, which normally gives
Fig. 2. Adult male tail phenotypes of mab-5 mutants, ventral view
except where noted. wt, wild type; hypo, mab-5(bx54)/mab-5(bx54),
lateral view (rays 1-3 lost, ray 4 separated away from rays 5 and 6
and extending to fan margin [arrowhead]); 0/+, mab-5(e1239)/+ (ray
4 extended to margin and fused to ray 3 on right side [arrowhead]);
gf, mab-5(e1751)/mab-5(e1751) (ray 1 fused to ray 2 [arrowheads];
ray 3 fused to ray 4 [upper open arrowhead], or at normal
anteroposterior position but not extending to fan margin [lower open
arrowhead]). Nomarski photomicrographs, scale bar indicates 10 µm.
rise to ray 4 (Fig. 1). Thus in the mab-5(bx54) mutant background the ray generated by V6.pppap has moved to a position
adjacent to the cloaca and extended to the margin of the fan; i.e.,
it has assumed the morphology of ray 3. This transformation was
not the consequence of the loss of rays 1, 2, and 3, because these
rays may be eliminated by cell ablation without a morphological transformation of ray 4 (Table 4, lines 21 and 22).
We also examined the effect of lowered mab-5 gene dosage
in a mab-5(0)/mab-5(+) heterozygote. In nematodes carrying a
single copy of the putative mab-5 null allele, e1239, the fourth
ray formed more anteriorly, opened at the margin of the fan,
and was fused to ray 3 in a small but significant percentage of
sides (11.5%) (Fig. 2; Table 2, line 10). (As there was no
evidence to the contrary, rays on the two sides of single animals
are assumed to be independent; thus data throughout is reported
as percentage of sides with a particular phenotype.) Fusion with
ray 3 suggests that the fourth ray now not only extends to the
fan margin at a more anterior position, but also expresses one
or more cell recognition molecules in common with ray 3. Thus,
when mab-5 function is decreased, several characteristics of the
fourth ray undergo a posterior-to-anterior transformation. We
refer to these several changes collectively as a change in the
identity of the ray generated by V6.pppap. As the ray normally
generated by this cell is referred to as ray 4, and since the new
identity appears to be the same as that normally taken by ray 3,
we refer to the phenotype in a mab-5 hypomorph or heterozygote as a transformation of the identity of ray 4 to that of ray
3. A similar shorthand is used throughout in referring to transformations affecting the other rays.
Increased mab-5 function results in transformation
of the identities of rays 1 and 3
A gain-of-function allele of mab-5 resulted in an anterior-to-
2584 K. L. Chow and S. W. Emmons
posterior transformation in ray identities opposite to that seen
with mab-5 loss-of-function alleles. mab-5(e1751) is a tandem
duplication covering the mab-5 gene that results in increased
or ectopic expression of normal mab-5 gene product
(Hedgecock et al., 1987; Salser and Kenyon, 1992; Salser et
al., 1993). In males homozygous for mab-5(e1751), ray 1 was
located more posteriorly, opened on the ventral surface of the
fan or at the fan margin, and fused with ray 2 (74%, n = 200).
Likewise, ray 3 was located more posteriorly, opened on the
ventral surface of the fan (80%, n = 200), and sometimes (3%,
n = 200) fused with ray 4 (Fig. 2). We interpret these morphological changes as anterior-to-posterior transformations in
the identities of ray 1 to that of ray 2, and ray 3 to that of ray
4.
mab-5 is not the sole determinant of ray identity
The results described above suggest that increased levels of
mab-5 gene expression caused rays 1 and 3 to develop like their
posterior neighbors, and decreased level of mab-5 gene
expression caused ray 4 to develop like its anterior neighbor.
This might be explained by the presence in the wild type of a
gradient of mab-5 gene activity through the tail region, with
increasing levels of activity determining more posterior ray
development. In a simple model of this kind in which mab-5
alone plays a determining role, if mab-5 gene function were
brought to its lowest level, or eliminated, all rays should take
on a common anterior morphology.
It was possible to test this prediction in a background containing a mutation at the lin-22 locus. lin-22 mutations have a
phenotype opposite to that of mab-5 mutations. That is,
whereas in mab-5 mutants the ray sublineage is not expressed
by the V5 and V6 lineages, in males homozygous for lin-22
loss-of-function mutations, the ray sublineage is expressed by
anterior seam cells in addition to V5, V6, and T (Horvitz et al.,
1983). In lin-22;mab-5 double mutants, some seam cells
express the ray sublineage, but these are not confined to the
tail region (Kenyon, 1986). This indicates that the combined
function of mab-5 and lin-22 is to regulate the spatial
expression of the ray sublineage. In mab-5;lin-22 double
mutants, some V-rays as well as T-rays are present in the fan.
We examined the morphology of such rays in a mab5(e1239);lin-22(mu2) double mutant. We found V-rays of all
types were generated at comparable, low frequencies (percentage of sides with a given ray was as follows: ray 1, 6%;
ray 2, 13%; ray 3, 17%; ray 4, 12%; ray 5, 15%; ray 6, 15%;
n = 200). Thus, there is no simple relationship between mab5 expression level and any one particular ray identity or morphology. Since posterior V-ray morphologies can develop even
in the absence of mab-5 gene function, additional genetic
functions must play a role in specification of ray morphology.
The HOM-C/Hox gene egl-5 is required for
specification of the identities of rays generated by
the V6 lineage
The AbdB-homolog egl-5 is required for expression of specialized cell fates in the region of the cloaca and elsewhere
(Chisholm, 1991). In males homozygous for egl-5(lf)
mutations, the morphology of the tail is severely affected by
transformations in the fates of several male-specific blast cells.
Among these, V6.ppppa is transformed into a hypodermal cell
and hence ray 6 is lost. Thus egl-5 plays a restricted role in
Fig. 3. Adult male tail phenotypes of egl-5 mutants. 0/0, egl5(n486)/egl-5(n486) lateral view (open arrowhead: clustered papillae
of rays 2-5 [arrowhead], papilla of ray 1 in normal position); 0/+,
egl-5(n486)/+ ventral view (open arrowhead: normal ray 6 absent;
arrowhead: ray 6 fused to ray 4 at position of ray 4). The right-hand
side is unaffected. Nomarski photomicrographs, scale bar indicates
10 µm.
specifying expression of the ray sublineage by a single branch
of the V6 lineage (Chisholm, 1991).
In addition, in egl-5 mutants, rays 2 through 5 appear to
loose their separate identities. During the late L4 larval stage,
papillae of rays 2 through 5 cluster together in the lateral hypodermis, and after morphogenesis short rays clustered and fused
together can be seen (Fig. 3). Fusion of rays 2 through 5 was
more dramatically demonstrated by Chisholm (1991), who
showed that in mosaic animals that were egl-5(−) in the V6
lineage but egl-5(+) elsewhere, all the rays were fully formed
and rays 2 through 5 were fused. These observations suggest
that in egl-5(−) rays 2 through 5 take on a single, common
identity. For example, they might assume some ground state
identity. Multiply fused rays usually extend to the fan margin,
suggesting the minimal ground state might be that of ray 3.
Ray 1 appeared to be unaffected (Fig. 3). We conclude that, in
addition to being required for expression of the ray 6 sublineage, egl-5 is required for specification of at least three of the
four identities taken by rays 2 through 5.
As was the case with mab-5, egl-5 is weakly haplo-insufficient for expression of the identity of one of the rays. In an egl5(0)/egl-5(+) heterozygote, in 7% of sides the morphology of
ray 6 was transformed into that of ray 4 (Table 2, line 4). In
these animals, ray 6 lost its distinctive, tapered morphology,
moved anteriorly, and fused with ray 4 (Fig. 3). In fused rays,
there were two ray openings, showing that the tip of ray 6,
which is not open in wild type, was transformed to one typical
of the other rays (Chow, Hall and Emmons, unpublished data).
Specification of ray 6 identity therefore appears to be sensitive
to the level of egl-5 gene activity.
Ray identity is sensitive to the relative numbers of
mab-5 and egl-5 gene copies
In other systems HOM-C/Hox genes may act redundantly,
combinatorially, or one may suppress the effects of another in
the establishment of a positional code (McGinnis and
Krumlauf, 1992; Morgan et al., 1992; Salser et al., 1993). To
test whether mab-5 and egl-5 interact in specification of ray
morphological identities, we analyzed strains carrying
mutations in both of these genes. Since the phenotype of the
mab-5 egl-5 double homozygote reflected the additive phenotypes of both mutations (absence of V-rays and deformed tail
region; data not shown), it was necessary to examine the inter-
Role of HOM-C/Hox genes in morphogenesis 2585
action of these genes in double heterozygotes. We found that
both genes have an effect on the specification of both ray 4 and
ray 6 morphological identities, and that the ratio of the number
of gene copies appears to be a critical factor.
The data for ray 6 are presented in Table 2, lines 1-4, 7, and
8. As already discussed, ray 6 morphology appeared to be
sensitive to the level of egl-5 gene product; in an egl-5(0)/egl5(+) heterozygote, ray 6 lost its distinctive morphology and
fused with ray 4 at a frequency of 7% (Table 2, line 4). In such
a heterozygote the ratio of mab-5(+) to egl-5(+) genes was 2:1.
In order to examine whether mab-5 also played a role in specification of ray 6, this ratio was increased by the introduction
of a single copy of the dominant mab-5 tandem duplication
allele e1751. In this background, the frequency of transformation of ray 6 to ray 4 was increased to 13% (Table 2, line 2).
The ratio of mab-5 to egl-5 was also increased by taking
advantage of the free duplication qDp3, which covers the
chromosomal region containing the entire C. elegans HOMC/Hox gene cluster, including mab-5 and egl-5. A ratio of mab5(+) to egl-5(+) of 3:1 was obtained by the introduction of
qDp3 into a strain homozygous for a strong egl-5 loss-offunction allele. In this background the frequency of transformation of ray 6 to ray 4 rose to 34% (Table 2, line 1). Evidence
that this increase was due at least in part to the additional copy
of mab-5(+) on qDp3 was obtained by analyzing a similar
qDp3-containing, egl-5(−) strain in which one chromosomal
wild-type copy of mab-5 was replaced by a mab-5 null allele.
In this strain the ratio of mab-5(+) to egl-5(+) was 2:1 and the
frequency of transformation of ray 6 to ray 4 was reduced to
16% (Table 2, line 3).
These results demonstrated that additional copies of mab5(+) enhanced the effect on ray 6 of lowering the number of
copies of egl-5(+). In a converse manner, lowering the number
of copies of mab-5(+) suppressed the effect of lowering the
number of copies of egl-5(+). In the double heterozygote (ratio
of mab-5(+) to egl-5(+) of 1:1), there was no detectable transformation of ray 6 to ray 4 (Table 2, lines 7 and 8). Thus it is
not the decreased number of egl-5 gene copies per se that
causes the ray 6 phenotype, but rather it is the number of egl5 gene copies relative to the number of mab-5 gene copies that
is important.
A similar result was obtained showing an effect of egl-5 in
specification of the morphology of ray 4 (Table 2, lines 7-12).
As described above, reduction of the number of wild-type
copies of mab-5 from two to one resulted in fusion of ray 4
with ray 3 at a frequency of 12% (Table 2, line 10). This effect
of reducing mab-5 gene number was partially suppressed if the
number of copies of egl-5 was also reduced (Table 2, line 8;
5.7% transformation of ray 4 to ray 3). Parallel results were
obtained with the mab-5 hypomorphic allele bx54 (5.8% transformation in a heterozygote [Table 2, line 9], 1.3% transformation in a double heterozygote with egl-5 [Table 2, line 7]).
When qDp3 was added to a strain homozygous for a mab-5
null allele (ratio of mab-5(+) to egl-5(+) of 1:3) the frequency
of transformation of ray 4 to ray 3 rose to 62% (Table 2, line
12). If one chromosomal egl-5(+) gene copy was replaced by
egl-5(n945) in such a strain (ratio of mab-5(+)/egl-5(+) of
1:2), the frequency of transformation dropped to 30.5%, indicating that part of the effect of qDp3 was due to the increased
dosage of egl-5(+) (Table 2, line 11).
Thus it appears that mab-5 and egl-5 both play roles in the
specification of both ray 4 and ray 6 morphological identities.
Their interaction is reciprocal: ray 6 is transformed with
increasing frequency to ray 4 as the ratio of egl-5(+) to mab5(+) falls below 1, and ray 4 is transformed with increasing
frequency to ray 3 as the ratio of mab-5(+) to egl-5(+) falls
below 1. Thus the ratio of mab-5 to egl-5 function may
normally help to differentiate these structures. Other rays may
not be affected in these experiments because the levels of mab5 and egl-5 gene activity examined do not cross critical thresholds for these rays.
Additional genes that affect ray identities are
dominant modifiers of HOM-C/Hox gene effects
We described previously a set of six genes required for development of the wild-type ray pattern (Baird et al., 1991).
Mutations in these genes caused transformations in ray morphology and fusion of rays that were interpreted to have
resulted from failed specification or implementation of distinct
ray morphological identities. For three of these genes, mab-18,
mab-20, and mab-21, the mutant phenotypes were similar to
those described here for mab-5 and egl-5. We have isolated an
additional mutation defining a new gene, mab-26, also with a
fused-ray phenotype. We asked whether these four genes acted
in the same pathway as mab-5 and egl-5 in specification of ray
morphology, by examining double mutants for evidence of
genetic interactions. Enhancement or suppression of HOMC/Hox gene effects would suggest action in the regulatory
pathway specifying ray identity. Absence of a synergistic interaction would suggest action independent of mab-5 and egl-5.
Two of the four genes have mutant phenotypes similar to
that of egl-5. Mutations in mab-18 and mab-21 result in loss
of the ray 6 morphology and fusion of this ray with ray 4.
Mutations in both genes are recessive or show very weak
dominant effects (Table 2, lines 14, 19-21) and are fully
penetrant. mab-18 encodes a putative transcription factor containing a homeodomain most closely related to that of vertebrate Pax-6 (Y. Zhang and S.W. Emmons, unpublished data).
Mutations in mab-21, in addition to affecting ray 6, also result
in the transformation of one seam hypodermal cell into a ray
precursor blast cell, which divides and gives rise to a tenth ray.
This suggests that mab-21 is a regulatory gene required for
specification of seam cell fates (K.L. Chow and S.W. Emmons,
unpublished data). mab-21 encodes a novel protein which also
has an essential embryonic function (K.L. Chow and S.W.
Emmons, unpublished data).
Mutations in both mab-18 and mab-21 were dominant
enhancers of egl-5. Reducing the number of copies of mab18(+) or mab-21(+) from 2 to 1 in an egl-5(0)/egl-5(+) heterozygote increased the frequency of transformation of ray 6
to ray 4 from 7% to around 30% (Table 2, compare lines 4, 13,
and 18, also compare lines 2 and 17). Since the mab-21 and
mab-18 alleles may not be null, these figures may underestimate the extent of the enhancement. Neither mutation interacted significantly with mab-5. There was little or no effect of
making these genes heterozygous in a mab-5(0)/mab-5(+) heterozygote (Table 2, compare lines 10, 16 and 25, compare lines
9 and 24). In the mab-18 mab-21 double heterozygote there
may have been a weak expression of the ray 6 to ray 4 transformation phenotype (Table 2, compare lines 14, 15, and 19).
We conclude that egl-5, mab-18, and mab-21 lie on a common
genetic pathway leading to expression of the characteristic
2586 K. L. Chow and S. W. Emmons
Table 3. Effect of mab-5 on frequency of ray fusions (%) in mab-20 and mab-26 mutants
ray
1
2
3
4
5
genotype
1
2
3
4
5
6
7
8
9
n
mab-20(bx61ts)/mab-20(bx61ts)
mab-20(bx61ts)/mab-20(bx61ts);mab-5(e1239)/+
mab-26(bx80)/mab-26(bx80)
mab-26(bx80)/+
mab-5(e1239)/+;mab-26(bx80)/+
9
35
78
0
5
15
67
95
0
8
67
99
98
25
74
64
99
89
25
74
1
1
30
0
0
1
1
49
0
0
2
0
34
0
n.d.
30
27
98
35
n.d.
30
27
98
35
n.d.
200
200
200
174
257
Animals containing mab-20(bx61ts) were scored at 25°C. Increased temperature had no effect on the penetrance of the mab-5(e1239)/+ phenotype (Table 2,
data not shown). Ray fusion partners were variable.
morphology of ray 6. The action of mab-18 and mab-21
appears to be either downstream of egl-5 or confined to cells
leading to ray 6, since if mutations in these genes acted in cells
leading to ray 4 either by lowering egl-5 activity or raising
mab-5 activity, suppression of the heterozygous effect of mab5 on ray 4 would have been expected.
The remaining two genes have mutant phenotypes affecting
several rays. The gene mab-20 is defined by one strong (bx24)
and one weak (bx61ts) allele (Baird et al., 1991). The strong
allele causes variable fusions of many rays, whereas in
homozygotes for the weak allele the most frequent fusion was
of ray 4 to ray 3, similar to the transformation seen in a mab5 heterozygote (Table 3, line 1). In the mab-20/+;mab-5/+
double heterozygote containing the strong mab-20 allele, there
was no enhancement of the mab-5 effect on ray 4 (Table 2,
compare lines 10 and 28), and a possible suppression of the
effect of egl-5 on ray 6 (Table 2, compare lines 4 and 26). In
a background homozygous for the weak mab-20 allele, introduction of a single mab-5 null mutation resulted in a significant increase in the frequency of fusions involving rays 1
through 4 (Table 3, compare lines 1 and 2). Thus mutations in
mab-5 and mab-20 are mutually enhancing, and this suggests
that mab-20 and mab-5 have related functions in specifying ray
identities. One possibility is that mab-20 mutations result in
lowered activity of the mab-5 gene in the ray identity pathway.
In a mab-26(bx80) homozygote, fusions of many rays occur
at high frequency (Table 3, line 3). bx80 is semidominant,
causing 25% transformation of ray 4 to ray 3 in heterozygotes
(Table 3, line 4). In the absence of further information about
the nature of the bx80 mutation and the mab-26 loss-offunction phenotype, it is not possible to infer a role of the wild
type mab-26 gene in specification of ray morphology. Nevertheless, interaction between the bx80 mutation and mutations
in mab-5 and egl-5 indicates that this mutation affects a process
associated with the action of these genes.
As for mab-20, the data for mab-26(bx80) suggest that this
mutation might lower the activity of mab-5. In the mab-5;mab26(bx80) double heterozygote, the frequency of transformation
of ray 4 to ray 3 was increased to 74% (Table 2, line 31),
whereas in the egl-5;mab-26(bx80) double heterozygote it was
reduced to 5% (Table 2, line 29). (On the assumption of independent action, the frequency of transformation of ray 4 to ray
3 in the mab-5;mab-26 double heterozygote was expected to
be 33%). In the egl-5;mab-26(bx80) double heterozygote the
frequency of transformation of ray 6 to ray 4 was decreased
from 7% to 0% (Table 2, compare lines 5 and 29).
In all of these phenotypic effects on ray fusion, mab26(bx80) and mab-5 null mutations have similar effects in heterozygotes, with mab-26(bx80) having the stronger effect.
Hence, one possible explanation for the mab-26(bx80)
phenotype was that this mutation decreased mab-5 gene
expression. However, with respect to expression of the ray sublineage, mab-26(bx80) differs from mab-5 null mutations: in a
mab-5(0) homozygote all the V-rays are lost, whereas there is
little or no ray loss in a mab-26(bx80) homozygote. Thus if the
effect of mab-26(bx80) is to decrease mab-5 gene expression,
it does so in a way that only affects specification of ray morphology. Alternative possibilities are that mab-26(bx80) acts at
the same step or downstream of mab-5 in a ray identity
pathway, or acts in an independent, parallel pathway.
Genetic functions that act within seam cells to
determine ray identities act cell-autonomously
within individual ray lineages
The transformations in ray morphology observed in the various
mutant backgrounds described above could be the result of
altered gene function in seam cells or in cells outside the seam.
In the next section we present evidence against gene action
outside the seam. Within the seam, these genes could act in
one or more of the terminal cells generated by the ray sublineage, in the ray precursor cells (Rn cells), or in cells at earlier
stages in the seam lineages. We conclude that the important
sites of altered gene action in these mutants lie within cells that
contribute to or generate only single rays. We reach this conclusion in part by considering the nature of the ray transformations seen in mutants. If the function of ray identity genes
was to determine the fates or potentialities of cells ancestral to
multiple rays, mutations would be expected to transform a
subset of adjacent, lineally related rays into a subset of rays
characteristic of a different lineage branch. However, the ray
transformations observed were not of this sort. Instead, single
rays of one branch were transformed into a ray descended from
a different lineage branch, while other rays of the first branch
remained unaffected. Thus, in mab-5 mutants, transformations
affected ray 3, descended from V6.pap, and ray 4, descended
from V6.ppp, while other rays generated by these precursor
cells (rays 2, 5, and 6) were unchanged. Thus these transformations cannot be the result of transformation of V6.pap into
V6.ppp, or vice versa. In the egl-5(lf) background ray 6,
descended from V6.pppp, was transformed to ray 4, normally
a descendant of V6.pppa. However, ray 5, also descended from
V6.pppp, was not affected. Thus this phenotype is not the result
of transformation of V6.pppp to V6.pppa. These transformations are most easily explained as transformations of terminal
branches of the lineage, and hence suggest that mab-5, egl-5,
and their genetic modifiers act within the terminal branches.
We investigated whether gene function critical for specifying ray morphology is cell-autonomous within the terminal
Role of HOM-C/Hox genes in morphogenesis 2587
Table 4. Morphological identities of rays generated after
laser ablation
Cell ablated
Late ablations
1 V5.pppp
2 V5.ppppp (R1)
3 V6.papa
4 V6.papp
5 V6.papp + V5.pppp
6 V6.pppa
7 V6.pppp
8 V6.papap (R2)
9 V6.pappp (R3)
10 V6.pppap (R4)
11 V6.ppppa (R6)
12 V6.ppppp (R5)
Expected
ray loss
1
1
2
3
1,3
4
5,6
2
3
4
6
5
Early ablations giving regulation
13 V6.p*
2,3,4,5,6
14 V6.p†
2,3,4,5,6
15 V6.p†
2,3,4,5,6
16 V6.pa + V6.pp
2,3,4,5,6
Early ablations giving no regulation
17 V6.pa
2,3
18 V6.pp
4,5,6
19 V6.pap + V6.ppp
2,3,4,5,6
20 V6.pap + V6.ppp
2,3,4,5,6
21 V5.ppp + V6.pap
1,2,3
22 V5.pppp + V5.pppa 1,2,3
+ V6.pap
23 V5.ppp + V6.ppp
1,4,5,6
24 V6.ppp + V6.ppa
4,5,6
25 V6.pap
2,3
26 V6.ppp
4,5,6
Observed
V rays
Number of
Lineages
experiments determined
2,3,4,5,6
2,3,4,5,6
1,3,4,5,6
1,2,4,5,6
2,4,5,6
1,2,3,5,6
1,2,3,4
1,3,4,5,6
1,2,4,5,6
1,2,3,5,6
1,2,3,4,5
1,2,3,4,6
1
5
3
6
1
19
4
9
14
18
5
19
2,3,4,5,6
1,2,3
2,3
2,3
1
1
3
1
1,4,5,6
1,2,3
1
none
4,5,6
4,5,6
5
3
1
2
1
1
2,3
1,2,3
1,4,5,6
1,2,3
3
1
4
3
*Ablated in early L1.
†Ablated in late L1; in #14 r1 was generated by V4.ppppp.
1
1
1
1
1
branches leading to single rays. In some systems, pattern
formation genes act cell-nonautonomously to generate signals
from one cell that influence the fates of neighboring cells
(Greenwald and Rubin, 1992). In order to determine whether
such signals pass between seam cells, we analyzed the effects
of laser ablations of ray precursor cells and their parents (Table
4, lines 1-12). We found it was possible to eliminate ray
precursor cells or their parents in all combinations without
altering the morphology of the remaining rays. The morphology of the ray generated by a given branch of the lineage was
independent of the morphology of neighboring rays. Similar
results have been reported previously (Sulston and Horvitz,
1977; Sulston and White, 1980). This indicates that for the
terminal branches of the ray lineages, defined as branches
giving rise to single rays, signals emanating from a ray of a
given type necessary for the fate of a neighboring ray are not
present. These results argue that the identity of each ray is
specified cell-autonomously in the terminal cells of the ray
lineages.
Evidence for a role of early seam cell signaling in
the determination of ray morphology
We considered two other modes of intercellular signaling that
might help to determine the properties of the cells in the ray
lineages. Cell signals might pass between seam cells early in
the lineage to restrict the potential of lineal precursors to
generate a subset of rays. Alternatively, positional signals
extrinsic to the seam might influence the fates of seam cells
later in the lineage.
We obtained evidence in favor of early seam cell signaling
and against extrinsic positional signals by carrying out
ablations of seam cells earlier in the lineage. Sulston and White
Fig. 4. Ray morphological identities after unilateral laser operation, and in a pal-1;lin-22 mutant. Adult male tails, ventral views. (A) wild type.
(B) Ablation of V6R.p (Table 4, line 13). Regulation of V5R.p generated rays 2-6. Ray 1 is absent. (C) Ablation of V6R.pa and V6R.pp (Table
4, line 16). Regulation of V5R.ppp generated rays 2 and 3 (arrowhead). The cell lineage is shown in Fig. 5A. (D) Ablation of V6L.ppp (Table
4, line 26). No regulation; rays 4-6 absent. The cell lineage is shown in Fig. 5B. Note that ray 3 is displaced posteriorly to the normal position
of ray 4 as a result of the deletion of rays 4-6, yet it remains extended to the margin (arrowhead). Likewise, ray 7 is displaced anteriorly, yet
remains open on the dorsal surface (open arrowhead). This illustrates that the position of a ray opening is an intrinsic property of a ray, and not
a consequence of mechanical or other factors associated with a particular anteroposterior location in the fan. (E) Ablation of V6L.pap (Table 4,
line 25). No regulation; rays 2 and 3 absent. (F) pal-1;lin-22 mutant. Rays 2 and 3 are generated on each side by V6 (open arrowheads). The
cell lineage is shown in Fig. 5C. Ray 3 is displaced posteriorly, and ray 7 is displaced anteriorly (arrowhead). Nomarski photomicrographs,
scale bar indicates 10 µm.
2588 K. L. Chow and S. W. Emmons
(1980) showed that ablation of V6 in early L1 resulted in a
transformation of the V5 lineage to one resembling that of V6.
We confirmed that such a transformed lineage generates rays
of the expected wild type morphologies (Fig. 4B; Table 4, line
13). Therefore regulation is complete, affecting both the
pattern of cell divisions as well as the morphological pattern
of the rays generated.
Because they generate a V6 lineage, such transformed V5
cells do not produce a postdeirid (Fig. 1A). We asked whether
regulation of the V5 lineage could occur at a later stage, after
V5.p was committed to produce a postdeirid. We found that
after ablation of V6.p late in the L1 larval stage (Table 4, lines
14 and 15), or V6.pp plus V6.pa early in the L2 larval stage
(Table 4, line 16), the fate of V5.ppp could be transformed to
one resembling V6.pap. That is, in these ablations, after all
seam cells of the V6 lineage branch were removed, V5.ppp
generated a lineage identical to that normally generated by
V6.pap, and gave rise to rays 2 and 3 (Figs 4C, 5A). Thus the
fate of V5.ppp is initially not determined, but is restricted to
generate ray 1 rather than rays 2 and 3 by the presence of more
posterior seam cells. Ablation of V6 lineage cells after division
of V6.pa and V6.pp resulted in no regulation of V5.ppp (Table
4, lines 19, 20, 24-26). Likewise, we observed no regulation
within the V6 lineage after early ablations (Table 4, lines 17,
18, 23, 25, and 26; Figs 4D,E, 5B).
In order to determine whether the effect of V6-lineage cells
on the fate of V5.ppp was to provide a signal acting on V5.ppp
to influence its fate, or was instead a passive effect on the
positions of V5.ppp or its descendants, thereby causing them
to receive certain seam-extrinsic positional cues and not others,
we followed the positions of cells in the V5.ppp lineage after
ablation of V6 cells. We found a lack of correlation between
the position of a cell and the type of ray it generated. After
ablation of cells of the V6 lineage, descendants of V5 moved
gradually to more posterior positions. In spite of the fact that
V5.ppppp reached the normal position of R4 or R5, it
generated ray 3 (Fig. 6). Likewise, V5.pppap generated ray 2
in spite of the fact that it occupied the normal position of R3.
Fig. 5. A V6.pap-like lineage can be executed by different cells.
(A) Generation of rays 2 and 3 from V5.ppp after ablation of V6.pa
and V6.pp. Animal shown in Fig. 4C. (B) Absence of regulation of
the V6.pap lineage after ablation of V6.ppp (arrow). Animal shown
in Fig. 4D. (C) Cell lineage in pal-1;lin-22. Animal shown in Fig. 4F.
Lineage nomenclature as in Fig. 1.
No hybrid lineages were observed. V5.ppp either generated ray
1 or rays 2 and 3; in no case did it generate ray 1 and ray 2, or
ray 3 and ray 4. These results are more consistent with a transformation in cell fate at the level of V5.ppp, affecting the type
of lineage and rays it could generate, than they are with the
presence of seam-extrinsic positional signals acting later on ray
precursor cells.
We were similarly able to show that the rays generated by
the normal precursors of rays 2 and 3 (R2 and R3) did not
depend on these cells occupying their normal positions along
the body. Although the lineage and rays generated by V6.pap
were not affected by ablation of V6.ppp (Table 4, line 26; Figs
4D, 5B), the positions of V6.pap descendants along the body
axis were affected. Thus, we observed V6.pappp (R3) in the
normal position of R4 or R5, yet its fate was not affected, and
it generated ray 3 as expected (Fig. 6).
Fig. 6. Lack of correlation between position of ray precursor cell and
type of ray generated. The relative positions of nuclei in the constant
interval defined by the positions of the V6.a nucleus and the anus
was judged by eye. Upper panel: The configuration of nuclei at the
time of ray precursor cell birth in wild type. Rn cell nuclei are in
bold, V6.a nucleus and anus (open arrowhead) are indicated for
orientation and comparison with Fig. 1B, which is several hours
later. Middle panel: The positions are shown of the ray precursor
cells that generated ray 2 (denoted R2) and ray 3 (denoted R3) under
three conditions. Movement of Rn cell nuclei from time of birth
(circle) to division (arrowhead) is indicated. Top three rows, wild
type; middle two rows, the V6R.pa and V6R.pp-ablated animal
shown in Figs 4C and 5A; bottom two rows, the V6L.ppp-ablated
animal shown in Figs 4D and 5B. Bottom panel: Positions of
V6.ppppp nuclei in 45 pal-1;lin-22 animals. Animals were not staged
and were scored once; hence the distribution should represent the
range of movement of this nucleus. This cell invariably generated ray
3.
Role of HOM-C/Hox genes in morphogenesis 2589
In order to test further whether ray morphology was determined by signals early or late in the lineage, we sought a
genetic method of uncoupling lineage, cell position, and ray
morphology. The genes pal-1 and lin-22 affect the response of
V6 and other seam cells to intercellular signaling (Waring et
al., 1992). In pal-1;lin-22 double mutants, V6 generates a
novel lineage giving rise to two rays (Waring and Kenyon,
1990) (Fig. 5C). We found that these two rays were invariably
rays 2 and 3 (Fig. 4F). This was in spite of the fact that the two
ray precursor cells were posterior of the positions normally
occupied by R2 and R3 (Fig. 6). This observation is consistent
with the interpretation that in the pal-1;lin-22 background, V6
or V6.p is restricted to a fate that can generate rays 2 and 3
only. It is not consistent with the presence of seam-extrinsic
positional cues acting later to determine the type of ray
generated by each lineage branch, unless such positional cues
were also affected by pal-1 and lin-22 mutations.
DISCUSSION
A role of HOM-C/Hox genes in ray pattern formation
The sensory rays of the C. elegans male tail have distinct morphogenetic properties. As a result, in the adult male the rays
are arrayed at reproducible positions within the acellular fan.
We have shown that the pattern formation mechanism that
guides the generation of ray differences depends in part on the
action of the HOM-C/Hox genes mab-5 and egl-5. It appears
that the level of activity of these two genes in the terminal
branches of the ray lineages is one of the critical factors that
determines what type of ray develops from each branch. These
results present the opportunity to study in greater detail the
steps in the morphogenetic pathway, including the mechanisms
that set the level of mab-5 and egl-5 gene activity within a
developmental field, the way in which mab-5 and egl-5 activity
specifies a positional value or developmental fate within the
field, the nature of additional upstream and downstream regulatory functions essential to this process, and the identities of
regulated downstream genes that execute the morphogenetic
program.
We present several lines of evidence that the site of action
of mab-5 and egl-5 is within the terminal branches of the seam
lineages, and that ray morphological identities are
autonomously determined within these branches. First, ray
transformations in mab-5 and egl-5 mutants were most simply
interpreted as transformations of one terminal branch into
another, suggesting the level of gene activity was critical
within each branch. Secondly, ablations of seam cells after the
mid L2 larval stage resulted in no regulation. As any ray type
could develop in the absence of any other ray type, this result
indicated an absence of signals emanating from a ray of one
type to determine the fate of another ray. Thus determination
of multiple ray types differs from determination of multiple
cell types in the Drosophila ommatidium, and the terminal
branches of the ray lineages do not constitute an equivalence
group (Greenwald and Rubin, 1992). Thirdly, there was no correlation between the position of a ray precursor cell and the
morphology of the ray it generated, suggesting an absence of
essential positional signals emanating from outside the seam.
Sulston and White (1980) similarly could find no evidence for
essential morphogenetic signals from outside the seam. They
carried out an extensive series of ablations in search of an
‘organizer’ or ‘inducer’ in the male tail analogous to the anchor
cell inducer of the vulva (Kimble, 1981). Ablations of the
gonad, tail neurons, and male-specific blast cells in different
animals resulted in no evident effects on the rays. Much of the
above evidence is negative, and therefore must be treated with
caution. However, taken altogether the results were most consistent with a late, cell autonomous action of mab-5 and egl-5
in defining the morphology of the V-rays. The levels of activity
of these two genes within the terminal branches of the ray
lineages appears to convey a particular positional or lineal
value to each cell individually, and to control, in an appropriate manner, the expression of downstream target genes.
How is positional or lineal value specified by the levels of
activity of mab-5 and egl-5? Analysis of the effects of raising
and lowering the level of activity or the number of copies of
these genes suggests that in wild type their activity may be
graded through the region of the tail where the V5 and V6 rays
form. A gradient of mab-5 activity in the body has previously
been suggested to define the ray domain in the tail (Kenyon,
1986). A graded pattern of expression of MAB-5 protein has
been observed in ventral cord neurons (Salser et al., 1993), and
expression of MAB-5 protein in V6 has been reported (Austin
and Kenyon, 1994). Alternatively, it might be the requirement
for mab-5 or egl-5 activity that is graded. Thus, a different
component of the pathway might be present in a gradient, and
this results in a variable threshold for mab-5 or egl-5 activity.
The presence of two graded molecules might provide the
basis of a code of positional or lineal values determining the
pattern of the V-rays. Indeed, we provide evidence that the
ratio of the number of mab-5 and egl-5 gene copies influences
the specification of the ray 6 and ray 4 fates. Since, as we have
argued above, mab-5 and egl-5 probably act cell autonomously
within the terminal branches of the ray lineages, it is the ratio
of function of these two genes within the terminal ray 4 and
ray 6 lineages that determines cell fate. We examined males
mutant in a third HOM-C/Hox gene, lin-39, and found they
possessed normal rays (data not shown). A code resulting from
overlapping expression of HOM-C/Hox genes has been
proposed to determine the identities of digits in the vertebrate
limb (Morgan et al., 1992). Increase or decrease of the level of
activity of individual HOM-C/Hox genes resulted in anterior
or posterior transformations of digit identities similar to the
anterior and posterior transformations of ray identities demonstrated here. Future studies should reveal whether the mechanistic basis of a HOM-C/Hox code is similar in these two very
diverse systems.
The opposite effects of raising and lowering the number of
copies of mab-5 and egl-5 suggests the possibility of a crosscompetitive interaction between their gene products. Crosscompetition could be at the level of competition for binding to
common sequences in critical target promoters, which would
thereby become responsive to the ratio of nuclear concentrations of MAB-5 and EGL-5 proteins. Or cross-competition
could be due to the formation of inactive heterodimers. Alternatively, cross-competition could be at the level of expression
of the HOM-C/Hox genes themselves. mab-5 and egl-5 might
be mutually repressing, so that increasing the number of copies
of one of these genes results in lowered expression of the other.
In this model, promoters of target genes would not bind both
mab-5 and egl-5 gene products. Repression of mab-5 gene
2590 K. L. Chow and S. W. Emmons
expression by egl-5 in a ventral cord blast cell has been demonstrated (Salser et al., 1993). In other systems, there is considerable evidence for cross-regulatory interactions between
HOM-C/Hox genes (see McGinnis and Krumlauf, 1992).
It is helpful in determining the roles of regulatory genes to
examine the ground state of the system in the absence of gene
function. It appears that in the absence of egl-5 gene product,
two V-ray identities are specified, one taken by ray 1, and the
other taken by rays 2 through 5. This may represent the ground
state generated by the remaining functions, and indicates that
in the presence of all other gene functions, egl-5 has an
essential role in specifying the differences among rays 2
through 5.
As no V-rays are generated in a mab-5 null mutant, the
ground state in this background cannot be assessed. However,
we have found that rays of correct morphology can be
generated in the presumed absence of mab-5 gene product
when a mutation at the lin-22 locus is also present. This result
appears paradoxical in view of the effects of altered mab-5
gene function discussed above. One possibility is that the mab5 allele used (e1239), which contains an altered splice site
(Table 1), still allows some gene function, and this low level
is elevated significantly in the lin-22 background. This explanation seems unlikely, because the phenotype of the mab-5;lin22 double mutant does not resemble a mab-5 hypomorph.
Whereas in a mab-5 hypomorph there is a higher frequency of
loss of anterior V-rays than posterior V-rays, in the lin-22;mab5 double mutant all rays were lost with similar frequency. If
e1239 is in fact null, then the mab-5 gene product is not
essential for the specification of multiple ray morphologies in
a lin-22 mutant background.
Function of fused ray genes
Although a central role of transcriptional regulation by HOMC/Hox genes in the generation of biological pattern has been
implicated for over a decade, few genes that are HOM-C/Hox
transcriptional targets are known, apart from the HOM-C/Hox
genes themselves (Andrew and Scott, 1992). It was recognized
early that region-specific cell recognition and adhesion was one
crucial aspect of cell differentiation regulated by HOM-C/Hox
genes (Garcia-Bellido, 1968; Morata and Lawrence, 1975;
Gauger et al., 1985). Yet the mechanisms by which this is
brought about are largely unknown. Our results suggest that
differentiation of the rays in the C. elegans male tail may
provide a promising opportunity to study this problem. In the
specification of ray identities by mab-5 and egl-5, there is no
change in cell lineage or obvious change in cell type. Instead,
the morphogenetic properties of the rays, and most cogently
the recognition functions of their constituent cells, appear to
be altered. This raises the possibility that in the ray lineages
genes encoding cell recognition functions may be direct targets
of mab-5 and egl-5 transcriptional regulation.
We sought to determine whether genes we identified previously, as well as one new gene, acted in the same pathway as
mab-5 and egl-5 in specifying ray morphology, and thus might
be candidates for mab-5 and egl-5 target genes. Our approach
was to look for evidence of genetic interactions. Mutations in
mab-18, mab-20, mab-21 and mab-26 cause loss of distinct ray
identities, in some cases in patterns similar to mab-5 and egl5 mutations. We have isolated multiple, recessive alleles of
mab-18, mab-20 and mab-21 in screens of relatively small
numbers of worms (Baird et al., 1991). This argues that these
mutations are loss-of-function alleles, and on this basis we
conclude that these genes play a role in specification of ray
morphology in wild type. We are unable to draw such a conclusion for mab-26, based on the single semi-dominant allele.
As this mutation may cause ectopic or neomorphic gene
activity, the normal function of mab-26 could be unrelated to
morphogenesis of the rays. Resolution of this issue awaits the
isolation and study of additional alleles at this locus. In the discussion below, the general conclusions apply in the case of
mab-26 to the mab-26(bx80) mutation only.
We found that mutations in mab-18, mab-20, mab-21, and
mab-26 were enhancers or suppressors of the effects of mab5 and egl-5 mutations on rays. This suggests that these genes
do not act independently of mab-5 and egl-5 in specifying ray
morphology. The cellular milieu in which they act is altered
when mab-5 or egl-5 gene expression is lowered, indicating a
proximity in their function in the ray morphogenetic pathway.
These results are consistent with any of these genes being transcriptional targets of mab-5 or egl-5. However, other possibilities remain, including roles in regulation of HOM-C/Hox gene
expression, or as HOM-C/Hox cofactors (McGinnis and
Krumlauf, 1992). Some dosage-dependent modifiers of the
Drosophila HOM-C/Hox gene Antennapedia are known to
participate in regulation of HOM-C/Hox gene expression
(Kennison and Tamkun, 1988). Alternatively, mab-18, mab20, mab-21, and mab-26 might act in one or more parallel
pathways partially redundant with the one in which mab-5 and
egl-5 act.
The ray pattern formation mechanism
Pattern formation in seam cell lineages involves spatially
restricted generation of postdeirid, alae, rays, and types of rays.
It is possible that a general patterning mechanism governs the
differentiation of all these structures. Kenyon and coworkers
have studied the formation of alae, postdeirid, and rays, and
have identified several genes essential to specification of these
structures (Kenyon, 1986; Waring and Kenyon, 1990, 1991;
Waring et al., 1992; Austin and Kenyon, 1994). They have
proposed that multiple mechanisms contribute to generation of
the axial pattern of the seam cell lineages. First, a graded signal
of unknown nature extending along the entire length of the
body appears to influence the tendencies of seam cells to
generate rays versus alae. Second, signals between seam cells
refine the pattern by inducing or inhibiting certain developmental pathways. Third, a prepatterning of seam cells, brought
about by an unknown mechanism, affects their individual
responses to cell signals. These three mechanisms are postulated to influence the differentiation of seam cells in part by
regulating the cell-autonomous expression level of mab-5,
which is expressed in a gradient with the highest level in the
tail.
Our results on generation of the ray pattern are consistent
with these proposals. We suggest that gradients of mab-5 and
egl-5 are present in the tail region, and that the levels of
expression of these genes influences the pattern of ray types in
a cell-autonomous manner. We show that a signal from cells
of the V6 lineage determines the types of rays generated by
V5.ppp. Our evidence is against a role of seam-extrinsic positional signals in defining the final ray fates. Waring and
Kenyon (1990) likewise favored signaling between seam cells
Role of HOM-C/Hox genes in morphogenesis 2591
over seam-extrinsic positional signals in the ray versus alae
decisions of the V4 and V5 lineages.
While giving an indication of the nature of the ray pattern
formation mechanism, our results leave unanswered a number
of questions. For example, we cannot yet explain how the
complete pattern of V-rays is generated, and likewise do not
address the generation of multiple ray types by the T lineage.
We have described one additional gene, mab-19, that could
play a role in the T lineage similar to the roles of mab-5 and
egl-5 in the V lineages (Sutherlin and Emmons, 1994). Further,
we do not know whether the relative expression levels of mab5 and egl-5 provide all the information required to discriminate up to 6 V-ray types; nor do we understand how such an
expression pattern could accomplish this. How HOM-C/Hox
genes convey positional identities to cells in general is not
known. One possibility is that multiple transcription factor
concentrations dictate multiple cellular responses. This
property has been demonstrated for the product of the bicoid
gene in Drosophila, which is also a homeodomain-containing
transcriptional regulator (Driever and Nüsslein-Volhard,
1989). Alternatively, or in addition, combinations of transcription factors may give a cellular outcome different from
the sum of the single factors acting separately, thus generating
a code. Such a mechanism has been demonstrated for cell fate
determination in the C. elegans ventral cord (Clark et al., 1993;
Wang et al., 1993).
In other well-studied examples of cellular pattern formation,
multiple mechanisms are superimposed to generate a reproducible spatial pattern of cell fates (Greenwald and Rubin,
1992). For example, in generation of the C. elegans vulva, a
graded diffusable signal and two different neighbor inhibitory
signals participate in the specification of three cell types
(Horvitz and Sternberg, 1991). Likewise, in generation of the
various organs of the Drosophila peripheral nervous system,
gross patterns generated by one pattern formation system are
refined by nearest-neighbor signaling systems (Heitzler and
Simpson, 1991). Thus it seems likely that several mechanisms
will also be involved in specification of the ray pattern.
We have shown that the types of rays generated by V5.ppp
are determined by a signal from its posterior neighbors. We did
not observe regulation indicative of signaling between other
branches of the ray lineages, or at later times. It is possible that
for these later lineages segregation of lineage branches with
distinct potentialities occurs by a cell-autonomous mechanism.
However, a cell-autonomous mechanism begs the question as
to how asymmetric cell divisions come to be correctly oriented
within the body. It seems more likely to us that signals between
seam cells occur at all levels. In this case, we have simply been
unable to demonstrate their presence. Our results indicate that
after committing to produce a postdeirid, the V5 lineage is
capable of undergoing regulation with respect to ray type for
only a brief interval (on the order of one or two hours) (Table
4; data not shown). If essential signals pass between neighboring cells during a shorter interval than this, possibly immediately after cells are born, it may be impossible to prevent
their action by a cell ablation experiment.
We suggest that ubiquitous signaling between neighbors
within the seam results in the generation of non-equivalent
precursor cells at each point in the ray lineages. The levels of
activity of the HOM-C/Hox genes mab-5 and egl-5 are likely
to be at least one component of this non-equivalence. Partly as
a result of differing mab-5 and egl-5 activity levels, each
precursor cell is capable of generating a subset of rays, and this
capability is continuously segregated into daughter cells until
the unique single lineage branches are generated.
We are grateful to S. Baird, N. Baker, D. Fitch, D. Stein, and L.
Stephens for their comments on the manuscript, and to members of
the laboratory for helpful discussions. We thank L. Avery, A.
Chisholm and S. Salser for sending nematode strains. Additional
strains were received from the Caenorhabditis Genetic Center, which
is funded by the NIH National Center for Research Resources
(NCRR). K. L. C. was supported by a Martin Foundation Postdoctoral Fellowship. This work was supported by NIH grant R01
GM39353.
REFERENCES
Andrew, D. J. and Scott, M. P. (1992). Downstream of the homeotic genes.
New Biologist 4, 5-15.
Austin, J. and Kenyon, C. (1994). Cell contact regulates neuroblast formation
in the Caenorhabditis elegans lateral epidermis. Development 120, 313-324.
Avery, L. and Horvitz, H. R. (1987). A cell that dies during wild-type C.
elegans development can function as a neuron in a ced-3 mutant. Cell 51,
1071-1078.
Baird, S. E., Fitch, D. H. A., Kassem, I. and Emmons, S. W. (1991). Pattern
formation in the nematode epidermis: determination of the spatial
arrangement of peripheral sense organs in the C. elegans male tail.
Development 113, 515-526.
Brenner, S. (1974). The Genetics of Caenorhabditis elegans. Genetics 77, 7194.
Chisholm, A. (1991). Control of cell fate in the tail region of C. elegans by the
gene egl-5. Development 111, 921-932.
Clark, S. G., Chisholm, A. D. and Horvitz, H. R. (1993). Control of cell fates
in the central body region of C. elegans by the homeobox gene lin-39. Cell
74, 43-55.
Driever, W. and Nüsslein-Volhard, C. (1989). Determination of spatial
domains of zygotic gene expression in the Drosophila embryo by the bicoid
morphogen. Nature 340, 363-367.
Emmons, S. W. (1992). From cell fates to morphology: developmental
genetics of the C. elegans male tail. BioEssays 14, 309-316.
Garcia-Bellido, A. (1968). Cell affinities in antennal homoeotic mutants of
Drosophila melanogaster. Genetics 59, 487-499.
Gauger, A., Fehon, R. G. and Schubiger, G. (1985). Preferential binding of
imaginal disk cells to embryonic segments of Drosophila. Nature 313, 395397.
Greenwald, I. and Rubin, G. M. (1992). Making a difference: the role of cellcell interactions in establishing separate identities for equivalent cells. Cell
68, 271-281.
Hedgecock, E. M., Culotti, J. G., Hall, D. H. and Stern, B. D. (1987).
Genetics of cell and axon migrations in Caenorhabditis elegans.
Development 100, 365-382.
Heitzler, L. and Simpson, P. (1991). The choice of cell fate in the epidermis of
Drosophila. Cell 64, 1083-1092.
Hodgkin, J., Horvitz, H. R. and Brenner, S. (1979). Nondisjunction mutants
of the nematode Caneorhabditis elegans. Genetics 91, 67-94.
Hodgkin, J., Edgley, M., Riddle, D. L. and Albertson, D. G. (1988).
Appendix 4, Genetics. In The Nematode Caenorhabditis elegans (ed. W.
Wood), pp. 491-584. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory.
Horvitz, H. R. and Sternberg, P. W. (1991). Multiple intercellular signaling
systems control the development of the Caenorhabditis elegans vulva.
Nature 351, 535-541.
Horvitz, H. R., Sternberg, P. W., Greenwald, I. S., Fixsen, W. and Ellis, H.
M. (1983). Mutations that affect neural cell lineages and cell fates during the
development of Caenorhabditis elegans. Cold Spring Harbor Symp. Quant.
Biol. 48, 453-463.
Kennison, J. A. and Tamkun, J. W. (1988). Dosage-dependent modifiers of
Polycomb and Antennapedia mutations in Drosophila. Proc. Natl. Acad. Sci.
85, 8136-8140.
Kenyon, C. (1986). A gene involved in the development of the posterior body
region of C. elegans. Cell 46, 477-487.
2592 K. L. Chow and S. W. Emmons
Kimble, J. (1981). Alterations in cell lineage following laser ablation of cells in
the somatic gonad of Caenorhabditis elegans. Dev. Biol. 87, 286-300.
McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial
patterning. Cell 68, 283-302.
Morata, G. and Lawrence, P. A. (1975). Control of compartment
development by the engrailed gene in Drosophila. Nature 255, 614-617.
Morgan, B. A., Izpisua-Belmonte, J. -C., Duboule, D. and Tabin, C. (1992).
Targeted misexpression of Hox-4. 6 in the avian limb bud causes apparent
homeotic transformation. Nature 358, 236-239.
Salser, S. J. and Kenyon, C. (1992). Activation of a C. elegans Antennapedia
homologue in migrating cells controls their direction of migration. Nature
355, 255-258.
Salser, S. J., Loer, C. M. and Kenyon, C. (1993). Multiple HOM-C gene
interactions specify cell fates in the nematode central nervous system. Genes
Dev. 7, 1714-1724.
Sulston, J. and Hodgkin, J. (1988). In The Nematode Caenorhabditis elegans.
(ed. W. B. Wood), pp. 587-606. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press.
Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the
nematode Caenorhabditis elegans. Dev. Biol. 56, 111-156.
Sulston, J. E. and White, J. G. (1980). Regulation and cell autonomy during
postembryonic development of Caenorhabditis elegans. Dev. Biol. 78, 577597.
Sulston, J. E., Albertson, D. G. and Thomson, J. N. (1980). The
Caenorhabditis elegans male: Postembryonic development of nongonadal
structures. Dev. Biol. 78, 542-576.
Sutherlin, M. E. and Emmons, S. W. (1994) Selective lineage specification by
mab-19 during Caenorhabditis elegans male peripheral sense organ
development. Genetics (in press).
Trent, C., Purnell, B., Gavinski, S., Hageman, J., Chamblin, C. and Wood,
W. B. (1991) Sex specific transcriptional regulation of the C. elegans sexdetermining gene her-1. Mech. Dev. 34, 43-56.
Wang, B. B., Müller-Immergluck, M. M., Austin, J., Robinson, N. T.,
Chisholm, A. and Kenyon, C. (1993). A homeotic gene cluster patterns the
anteroposterior body axis of C. elegans. Cell 74, 29-42.
Waring, D. A. and Kenyon, C. (1990). Selective silencing of cell
communication influences anteroposterior pattern formation in C. elegans.
Cell 60, 123-131.
Waring, D. A. and Kenyon, C. (1991). Regulation of cellular responsiveness
to inductive signals in the developing C. elegans nervous system. Nature
350, 712-715.
Waring, D. A., Wrischnik, L. and Kenyon, C. (1992). Cell signals allow the
expression of a pre-existent neural pattern in C. elegans. Development 116,
457-466.
White, J. (1988). The anatomy. In The Nematode Caenorhabditis elegans. (ed.
W. Wood), pp. 81-122. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press.
Wood, W. B. (1988). The Nematode Caenorhabditis elegans. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory Press.
(Accepted 13 June 1994)