1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA 98109, USA
2 Howard Hughes Medical Institute, Seattle, WA 98109, USA
3 Department of Molecular Genetics, The Ohio State University, Columbus, OH
43210, USA
4 Department of Zoology, University of Washington, Seattle, WA 98195, USA
Author for correspondence (e-mail:
jpriess{at}fhcrc.org)
Accepted 22 January 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: C. elegans, GLP-1, Notch, T-box, Mesoderm, Induction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite the importance of Notch signaling, little is known about how
individual Notch-mediated interactions specify distinct cell fates at
different times and places in development. The receptors GLP-1/Notch and
LIN-12/Notch appear to be functionally equivalent, as are the ligands
expressed by the various signaling cells
(Fitzgerald et al., 1993;
Fitzgerald and Greenwald,
1995
; Gao and Kimble,
1995
; Moskowitz and Rothman,
1996
; Shelton and Bowerman,
1996
). Moreover, all known examples of Notch signal transduction
in C. elegans appear to involve a single transcriptional effector
called LAG-1/Suppressor of Hairless [Su(H)]
(Christensen et al., 1996
).
Thus, cell fate specificity must be achieved by factors that act in
combination with Notch signaling.
At least four distinct interactions occur during the first few cell
divisions of the C. elegans embryo, providing a relatively simple
experimental system to analyze a network of Notch-mediated cell fate decisions
(for a review, see Schnabel and Priess,
1997). The anterior cell in the two-cell stage embryo is called
AB, and all of the early descendants of AB express the receptors GLP-1/Notch
or LIN-12/Notch. Various AB descendants contact one of several
ligand-expressing cells that are born at different times and places during the
early divisions, and change their fate accordingly. In genetic studies of
Notch-mediated, binary cell fate decisions, one cell fate can often be
considered as `primary' and a second cell fate as `secondary'; Notch function
is required for the secondary, but not primary, fate
(Artavanis-Tsakonas et al.,
1999
). In the absence of all Notch-mediated interactions in C.
elegans embryos, AB descendants adopt highly patterned ectodermal fates
that will thus be described here as primary fates.
The first Notch interaction occurs at the four-cell stage when the
posterior daughter of AB, called ABp, contacts a cell called P2
that expresses a Notch ligand (see Fig.
1). The interaction between P2 and ABp causes the ABp
descendants to adopt new fates that we describe here as secondary fates; cells
with secondary fates remain ectodermal precursors, but have a pattern of
differentiation that is distinct from cells with primary fates. The anterior
daughter of AB, called ABa, does not contact P2 and thus produces
descendants that initially retain their potential for primary fates. At the
12-cell stage, however, two of the ABa granddaughters contact a new
ligand-expressing cell called MS. This second Notch interaction induces those
two ABa granddaughters to adopt novel, tertiary fates and become mesodermal
precursors. During the next few cell divisions, there are third and fourth
Notch interactions that further diversify the fates of some ABp descendants
(see below and legend to Fig.
1). Coincident with the Notch-mediated specification of cell
fates, a separate anteroposterior polarity system generates additional
differences between sister cells that are born from anteroposterior cell
divisions. Thus, there are two types of primary fates (1a and 1p) depending on
whether a cell is an anterior sister (1a) or posterior sister (1p;
Fig. 1B). Similarly, there are
two types of secondary fates and two tertiary fates
(Fig. 1B). These
anteroposterior differences appear to involve POP-1, a transcription factor
that is localized asymmetrically after all anteroposterior divisions of the AB
descendants (Kaletta et al.,
1997; Lin et al.,
1998
; Park and Priess,
2003
).
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Screens for Aph mutants
General procedures for isolating embryonic lethal mutants were as described
elsewhere (Page et al., 1997).
Mutations in tbx-38 were isolated by mutagenizing eDf20/qC1;
lin-2(e1309) animals. Candidate mutants were mated with dpy-18(e364)
spe-6(hc49)/qC1; lin-2(e1309); him-8(e1489) males. Recombinant
dpy-18(e364) tbx-38(mutant)/dpy-18(e364) spe-6(hc49) animals were
picked from the eDf20 tbx-38(mutant)/dpy-18(e364) spe-6(hc49)
heterozygotes and allowed to self. Mutations in tbx-37 were isolated
by mutagenizing dpy-18(e364)tbx-38(e460) homozygous animals, and were
then balanced with qC1. All tbx-37 mutant strains were
outcrossed two or more times using dpy-18(e364) spe-6()/qC1; lin-2(e1309);
him-8(e1489) males.
Mapping deficiencies and identifying mutations in Tbx genes
Genomic DNA was amplified from single embryos as described
(Muhlrad, 2002). To map
regions deleted in deficiencies, dead eggs were collected from the progeny of
deficiency heterozygotes and were used in single-embryo PCR reactions. Three
sets of primers were used for each mapping experiment: an experimental primer
set, a positive control set recognizing sequences on an intact chromosome, and
a negative control set targeting a sequence known to be removed by the
deficiency. Mutations within tbx-37 and tbx-38 were
identified by amplifying and sequencing DNA from single Aph. Sequencing
reactions were performed at the FHCRC core sequencing facility and were
repeated at least once for each identified mutation.
Transgene construction
Standard techniques were used to manipulate and amplify DNA. All transgene
constructs were made using PCR fusion techniques
(Hobert, 2002). To construct
tbx-37::gfp and tbx-38::gfp, promoters (434 bp or 423 bp
5' of the start codons, respectively) were amplified and fused to
gfp-coding sequences from plasmid pPD95.69 (1995 Fire lab vector kit,
www.ciwemb.edu).
In a second tbx-38 fusion construct, a sequence beginning 423 bp
5' of the start codon and including the entire 1838 bp coding sequence
was amplified and fused to gfp-coding sequence from the plasmid
pPD95.75 (1995 Fire lab vector kit,
www.ciwemb.edu).
Predicted start codons for TBX-37 and TBX-38 were obtained from the Wormbase
website (Wormbase web site,
http://www.wormbase.org).
Worm transformations
Purified yeast artificial chromosome Y47D3 was injected into
zuDf3/qC1 worms with a dominant rol-6
co-transformation marker as described (Mello and Fire, 1995). Wild-type worms
were injected with tbx-37::gfp, tbx-38::gfp or
tbx-38::tbx-38/gfp, and a dominant rol-6
co-transformation marker (Mello and Fire, 1995). The resulting
extrachromosomal arrays were integrated by gamma-irradiation (Mello and Fire,
1995).
Phenotypic and lineage analysis
One quarter of the self-progeny of tbx-37(zu467) dpy-18(e364)
tbx-38(zu460)/qC1 adult hermaphrodites are tbx-37
tbx-38 mutant embryos. In experiments on fixed and stained embryos,
mutant phenotypes were identified by examining all of the progeny. For
analysis of live, early cells, mutant embryos were identified retrospectively
after allowing each embryo to develop to terminal stage. For lineage analysis
of mutant embryos, the marker pha-4::gfp was crossed into the
parental strain. Embryos were selected for analysis that lacked GFP expression
in the early ABa descendants.
Antibodies
An artificial tbx-38 cDNA was constructed by fusing each of the
three predicted tbx-38 exons after amplification by PCR. The
tbx-38 `cDNA' was cloned into HindIII and XhoI
sites of the pET-21b His-tag protein expression vector (Novagen). His-tagged
TBX-38 was purified over a nickel column (QIAexpressionist kit, Qiagen) and
injected into mice at the FHCRC Hybridoma Production Facility as described
(Wayner and Carter, 1987).
Hybridoma supernatants were assayed by immunostaining early embryos. Fixation
and staining have previously been described
(Lin et al., 1995
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Embryos from each of the tbx-37 dpy-18 tbx-38 triple mutant
strains appeared essentially identical in the light microscope. The embryos
had a well-differentiated posterior half-pharynx that was enclosed by a
prominent basement membrane and that was attached to the intestine (bracketed
region in Fig. 3B). In
wild-type embryos, the apical surfaces of cells in the anterior and posterior
halves of the pharynx can be visualized by immunostaining for adherens
junctions (Fig. 3C). In the
mutant embryos there was a gap in the staining pattern where the anterior half
of the pharynx would normally form (Fig.
3D). A rectum was visible at the posterior end of the intestine in
most embryos (arrowheads, Fig.
3B,D). Embryos from all strains showed variable defects in body
elongation; either the embryos ruptured along their ventral midline without
elongating, or the embryos elongated into lumpy, mis-shapen worms (see below).
Previous studies have shown that embryos defective in the GLP-1/Notch
signaling pathway have duplications in right and left lateral skin cells
called seam cells (Priess et al.,
1987). We found defects in seam cell organization in all mutant
embryos examined (n>100). Extra seam cells were present along the
left lateral sides of almost all mutant embryos (compare
Fig. 3F with Fig. 3E); however,
the right lateral side either contained the normal number of seam cells or too
few seam cells (see below). For a detailed analysis of the mutant phenotypes,
we constructed the balanced strain tbx-37(zu467) dpy-18(e364)
tbx-38(zu460)/qC1 and examined embryos from these heterozygous
adults; the homozygous tbx-37(zu467) dpy-18(e364) tbx-38(zu460)
embryos are hereafter referred to as tbx-37 tbx-38 mutant
embryos.
|
|
In wild-type embryos, PHA-4 expression begins in the early descendants of
MS and ABa that are the precursors of pharyngeal cells
(Horner et al., 1998;
Kalb et al., 1998
). We
monitored PHA-4 expression in early embryogenesis with a pha-4::gfp
transgene (Alder et al., 2003
).
Control embryos from wild-type animals that contained the transgene first
showed PHA-4::GFP expression in the MS and ABa descendants at the 44-cell
stage, one cell cycle earlier than reported previously for PHA-4
immunostaining (Horner et al.,
1998
; Kalb et al.,
1998
). PHA-4::GFP was detected in the four granddaughters of MS,
and in the two anteriormost granddaughters of both ABalp and ABara (arrows in
Fig. 4D, see also
Fig. 1B). Unexpectedly, we
occasionally observed a low level of PHA-4::GFP expression in the anterior two
granddaughters of ABala (data not shown). ABala does not produce pharyngeal
cells and is not thought to be signaled by MS; however, ABala descendants show
transient contacts with the MS daughters. PHA-4::GFP expression did not
persist in the ABala descendants in subsequent cell cycles and was not
analyzed further.
We found that tbx-37 tbx-38 mutant embryos at the 44-cell stage had normal PHA-4::GFP expression in the four MS granddaughters, but did not show PHA-4::GFP expression in any ABa descendant (arrows in Fig. 4H). Similarly, PHA-4::GFP expression was not detected in any of several ABa descendants that were monitored intermittently over the next three or four cell cycles. Thus PHA-4 expression is not initiated in the ABa descendants of tbx-37 tbx-38 mutant embryos.
In addition to inducing PHA-4 expression, signaling from MS and GLP-1/Notch
signal transduction have a second role in repressing the expression of the
protein LAG-2 in ABa descendants
(Moskowitz and Rothman, 1996).
LAG-2 normally is expressed in the four granddaughters of ABala, a cell that
does not contact MS, but is repressed in the four granddaughters of ABara, a
cell that contacts MS (see Fig.
1B) (Moskowitz and Rothman,
1996
). When MS signaling or GLP-1/Notch signal transduction are
blocked, LAG-2 is expressed in each of the ABara granddaughters in addition to
the ABala granddaughters. To examine whether MS signaling occurs in tbx-37
tbx-38 mutant embryos, we crossed in a lag-2::gfp transgene
(Moskowitz and Rothman, 1996
).
Similar to wild-type embryos, the ABala granddaughters expressed LAG-2::GFP in
tbx-37 tbx-38 mutants, but the ABara granddaughters did not (16/16
embryos; arrows in Fig. 5A). To
confirm that MS signaling repressed LAG-2 expression in the ABara
granddaughters, we used a laser microbeam to kill MS. All of the self-progeny
of heterozygous tbx-37 tbx-38 /+ + hermaphrodites in which MS was
killed had LAG-2::GFP expression in each of the ABara and ABala granddaughters
(arrows in Fig. 5B; n=15/15). We conclude that MS signaling occurs in tbx-37
tbx-38 mutant embryos and represses LAG-2, but that MS signaling does not
result in PHA-4 expression.
|
|
In the fourth Notch interaction, certain descendants of the MS cell (MSapa
or MSapp) become new signaling cells that activate Notch in adjacent ABplp
descendants (Moskowitz and Rothman,
1996). We found that these ABplp descendants appeared to
differentiate normally in tbx-37 tbx-38 mutant embryos
(Table 1). For example, some of
these descendants became the excretory cell or formed the rectum, both of
which are visible in most tbx-37 tbx-38 mutant embryos
(Fig. 3B, arrowhead). Thus,
three out of the four early, Notch-mediated interactions appear to occur
normally in tbx-37 tbx-38 mutant embryos.
We next examined the cell lineages of several ABa descendants whose fates are determined by the second Notch interaction (signaling from MS to ABalp and ABara at the 12-cell stage; Fig. 1B). Although the development of ABalp was highly abnormal in the tbx-37 tbx-38 mutant embryos, the defects did not match the abnormalities seen when MS signaling is prevented (Table 1). For example, in wild-type embryos, ABalpppaaaa undergoes programmed cell death, and this same cell differentiates without dividing when MS signaling is prevented. However, this cell did not undergo programmed cell death or differentiate in the tbx-37 tbx-38 mutant embryos, and instead continued to divide. Thus, tbx-37 tbx-38 embryos have lineage defects in cell types normally specified by MS signaling, although the defects do not match those expected from a simple loss of Notch signal transduction.
To determine whether the tbx-37 and tbx-38 genes have a
role in the ABa descendants that are not signaled by MS, we examined the
development of ABarp (Fig. 1B).
In wild-type embryogenesis, each ABarp granddaughter generates a clone of
cells that is located on the dorsal surface of the embryo; most of these cells
will form the hypodermis or skin of the embryo
(Fig. 3G, see legend for color
code). In the tbx-37 tbx-38 mutant embryos, most of the descendants
of ABarpaa (magenta) and all of the descendants of ABarpap (green) ingressed
into the body cavity rather than remaining on the dorsal surface
(Fig. 3H). These abnormal cell
ingressions created transient gaps on the dorsal surface
(Fig. 3H, arrow). Similar gaps
occur only on the ventral surface of wild-type embryos, as cell ingressions
normally are restricted to the ventral surface (Nance and Priess, 2002). In
wild-type embryos, the clones generated from the sister cells ABarppa (blue)
and ABarppp (yellow) separate from each other to form left and right lines of
skin cells on the lateral surfaces of the body
(Fig. 3G) (Sulston et al., 1983). These
clones did not separate normally in any of three tbx-37 tbx-38
embryos analyzed (Fig. 3H);
this defect probably contributes to the abnormal numbers of seam cells
observed on the lateral sides of terminal tbx-37 tbx-38 embryos
(Fig. 3F). Although the sister
cells ABarppa and ABarppp normally have identical fates, only ABarppa was
markedly abnormal in tbx-37 tbx-38 embryos (see Discussion). In
conclusion, tbx-37 tbx-38 mutant embryos have defects in multiple ABa
descendants irrespective of whether or not these cells undergo the second
Notch interaction (MS signaling). By contrast, the tbx-37 tbx-38
embryos do not appear to have defects in any of the ABp descendants
examined.
TBX-37 and TBX-38 are expressed in ABa, but not ABp descendants
To assay TBX-37 and TBX-38 expression, a monoclonal antibody (mAbT38) was
generated against a full-length TBX-38 fusion protein, and tbx-7::gfp,
tbx-38::gfp and tbx-38::tbx-38/gfp transgenes were constructed
and integrated into chromosomes (Materials and methods). mAbT38 did not stain
tbx-37 tbx-38 mutant embryos (data not shown), but showed strong
staining of nuclei in 24-cell wild-type embryos
(Fig. 5C). At the 24-cell
stage, there are eight descendants of ABa and eight descendants of ABp; only
the eight ABa descendants expressed TBX-38
(Fig. 5C; cells identified in
Fig. 5D). Staining diminished
markedly in the ABa descendants during the next cell cycle, with very little
or no staining visible thereafter (data not shown). Similarly, the
tbx-37::gfp and tbx-38::gfp transgenic embryos had GFP
expression exclusively in the eight ABa descendants at the 24-cell stage
(Fig. 5E,G and data not shown).
Although antibody staining suggested that endogenous TBX-38 is an extremely
short-lived protein, TBX-38::GFP persisted in all of the ABa descendants
throughout most of embryogenesis (data not shown).
We asked whether signaling from MS induced the expression of the TBX-37, TBX-38 proteins in ABa descendants. Killing MS with a laser microbeam did not prevent TBX-38::GFP expression in any of the ABa descendants (8/8 experiments). Similarly, RNAi-mediated depletion of LAG-1, the transcriptional effector of GLP-1/Notch, did not alter the pattern of TBX-37::GFP expression in ABa descendants (27/27 embryos with terminal lag-1 phenotypes). Thus, MS signaling and Notch signal transduction are not required for TBX-37, TBX-38 expression in ABa descendants.
We wanted to determine whether SKN-1, a maternally provided transcription
factor, had a role in TBX-37, TBX-38 expression. SKN-1 is required for PHA-4
expression and mesoderm development, both through the Notch-dependent pathway
(anterior pharynx produced by ABa descendants) and Notch-independent pathway
(posterior pharynx produced by MS descendants)
(Bowerman et al., 1992). SKN-1
is present at high levels in MS, and at lower levels in ABa and ABa
descendants (Bowerman et al.,
1993
). We crossed the tbx-37::gfp transgene into a
skn-1(zu67) mutant strain and found that each of 17 skn-1
mutant embryos examined showed the wild-type pattern of GFP expression in ABa
descendants at the 24-cell stage. Thus, mutations in skn-1 do not
appear to prevent ABa descendants from producing mesoderm by blocking the
expression of TBX-37. This result is consistent with previous evidence that
SKN-1 does not regulate the competence of ABa descendants to respond to MS
signaling, but may instead regulate some aspect of MS signaling
(Shelton and Bowerman,
1996
).
We next asked whether TBX-37, TBX-38 was repressed in ABp descendants by signaling from P2. For this experiment, the tbx-37::gfp transgene was crossed into an apx-1(zu183) mutant strain; apx-1 encodes the P2 ligand for GLP-1/Notch. We found that all of the 24-cell embryos examined showed TBX-37::GFP in both ABa and ABp descendants (27/27 embryos; Fig. 5H). Similarly, both ABa and ABp descendants expressed TBX-37::GFP in many embryos where LAG-1 was depleted by RNAi (14/22 embryos; data not shown). Therefore TBX-37 expression is prevented in ABp descendants by P2 signaling and by GLP-1/Notch signal transduction.
Loss of tbx-37 tbx-38 (+) activity suppresses posterior defects in apx-1 mutants
In the absence of Notch-mediated interactions, ABp descendants adopt
primary fates instead of their normal secondary fates. We have shown here that
preventing the first Notch interaction causes ABp descendants to express
TBX-37, TBX-38 inappropriately. Moreover, TBX-37, TBX-38 functions appear to
contribute to primary fates, because the development of ABarp (primary fate
1p) is abnormal in tbx-37 tbx-38 mutant embryos
(Fig. 1B). These findings
together raise the possibility that the first Notch interaction might in part
permit normal ABp development by preventing TBX-37, TBX-38 expression. To test
this possibility, we constructed and analyzed apx-1; tbx-37 tbx-38
triple mutant embryos. In wild-type embryos, ABp descendants contribute
predominately to posterior body morphology, these descendants form tail
structures including the rectum, tail spike and ventral hypodermis
(Sulston et al., 1983).
apx-1 mutants defective in the first Notch interaction, and
glp-1 mutants defective in both the first and second Notch
interactions, do not undergo posterior body morphogenesis and lack each of
these posterior features (Fig.
6A) (Mello et al.,
1994
). Remarkably, we found that late stage apx-1; tbx-37
tbx-38 triple mutant embryos had a well-formed tail including a rectum
(Fig. 6B, large arrow) and a
tail spike (insert, Fig. 6B).
At earlier morphogenesis stages, these embryos had a group of ventral
hypodermal cells that appeared identical to the wild-type ABp descendants in
pattern and number (compare Fig. 6C with
6D). As expected, the heads of the triple mutant embryos contained
only a partial pharynx and had variable anterior defects similar to those of
the tbx-37 tbx-38 double mutant embryos. We did not perform a
detailed lineage analysis of the triple mutant embryos. However, the normal
appearance of posterior, ABp-derived structures argues that the first Notch
interaction is largely, if not entirely, dispensable if TBX-37, TBX-38
function(s) are prevented in ABp
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the second Notch interaction (MS signaling) induces cells to
become mesodermal precursors that form the pharynx, the first Notch
interaction (P2 signaling) prevents cells from becoming mesodermal
precursors. If the first Notch interaction does not occur, embryos have a
hyperinduction of pharyngeal tissue (Fig.
6A) (Hutter and Schnabel,
1994; Mello et al.,
1994
; Mango et al.,
1994a
; Moskowitz et al.,
1994
). In normal development, MS signaling induces ABa, but not
ABp, descendants to become mesodermal precursors (black arrows from MS in
Fig. 7A). However, MS and its
sister cell, called E, both have the ability to signal, and one or both of
these cells contact some ABp descendants in addition to contacting ABa
descendants (gray arrows from MS in Fig.
7A) (Lin et al.,
1998
). When P2 signaling is blocked, either by
physically removing P2 or by mutations in the P2 ligand
encoded by apx-1, MS and E induce these additional ABp descendants to
become mesodermal precursors (black arrows from MS in
Fig. 7B). We have shown that
mutations in apx-1 cause the inappropriate expression of TBX-37,
TBX-38 in ABp descendants. In addition, we have shown that removing TBX-37,
TBX-38 activities from apx-1 mutant embryos prevents the
hyperinduction of pharyngeal cells (Fig.
7C; Fig. 6B). Thus,
the competence of both ABa and ABp descendants to become mesodermal precursors
in response to the second Notch interaction is determined by the pattern of
expression of TBX-37, TBX-38.
|
Genetic studies have been reported on only two T-box family members in
C. elegans, mab-9 and mls-1
(Chisholm and Hodgkin, 1989;
Woollard and Hodgkin, 2000
;
Kostas and Fire, 2002
). MAB-9
is required to pattern the posterior hindgut; in mab-9 mutants,
posterior blast cells in the hindgut adopt characteristics of their anterior
neighbors (Chisholm and Hodgkin,
1989
). MLS-1 has a role in specifying muscle cell types; mutations
in mls-1 cause presumptive uterine muscle precursors to differentiate
as vulval muscles (Kostas and Fire,
2002
). The cell fate decisions mediated by MAB-9 and MLS-1 are not
known to involve Notch signaling directly, although Notch signaling has roles
both before and after the events that differentiate uterine from vulval
muscles.
An important task of future studies is the identification of the Notch
target(s) that function in conjunction with TBX-37, TBX-38 to activate PHA-4
expression. Signaling from MS begins sometime during the 12-cell stage.
Because the interval between the 12-cell and 24-cell stages is only 16
minutes, it is likely that Notch targets are transcribed and translated late
in the 12-cell stage or early in the 24-cell stage. TBX-37, TBX-38 are
expressed at the 24-cell stage, suggesting that TBX-37, TBX-38 may be
co-expressed with direct Notch targets. There are several possibilities for
how these proteins might interact directly or indirectly. Recent studies have
provided examples of T-box proteins that bind to other proteins to control
tissue differentiation. These partners include GATA transcription factors,
homeodomain proteins and the membrane-associated guanylate kinase CASK/LIN-2
(Garg et al., 2003;
Hiroi et al., 2001
;
Bruneau et al., 2001
).
CASK/LIN-2 can enter the nucleus and form a complex with the T-box protein
Tbr-1 to induce the transcription of target genes (Hsueh et al., 2000). LIN-2
is the C. elegans homolog of CASK/LIN-2. Mutations in the gene
encoding LIN-2 have no affect on embryonic viability, suggesting that LIN-2 is
not an essential co-factor for TBX-37, TBX-38. The mouse T-box protein Tbx5
can bind the homeodomain protein Nkx2-5 and recognize adjacent binding sites
within the promoter of a target gene. The C. elegans homolog of
Nkx2-5, which is encoded by ceh-22, is expressed in pharyngeal
muscles and the pharyngeal defects caused by a mutation in ceh-22 can
be rescued by a vertebrate nkx2.5 gene
(Okkema et al., 1997
;
Haun et al., 1998
). However,
CEH-22/Nkx2-5 expression occurs after, and is dependent on, PHA-4 expression
(Mango, 1994; Okkema et al., 1994), suggesting that CEH-22 is not likely to
interact with TBX-37, TBX-38.
Specification of ABp by the first Notch interaction
The first Notch interaction has been thought to `induce' the ABp fate;
P2 and the Notch ligand APX-1 are essential for normal ABp
development, and forcing P2 into ectopic contact with ABa causes
ABa descendants to inappropriately adopt ABp-like fates
(Hutter and Schnabel, 1994;
Mango et al., 1994a
;
Mello et al., 1994
;
Moskowitz et al., 1994
). Our
present study provides strong evidence that the first Notch interaction is
permissive rather than instructive for ABp development. TBX-37, TBX-38 are not
detectable in ABp descendants in wild-type embryos, and tbx-37 tbx-38
mutant embryos appear to have normal ABp development. Thus, TBX-37, TBX-38 do
not contribute to ABp development in wild-type embryogenesis. Preventing the
first Notch interaction causes ABp descendants to express TBX-37, TBX-38
inappropriately and to adopt incorrect fates. However, preventing both the
first Notch interaction and TBX-37, TBX-38 expression simultaneously allows
apparently normal differentiation of ABp descendants. Together, these results
indicate that the primary, if not sole, function of first Notch interaction is
to repress TBX-37, TBX-38 expression in ABp descendants. Notch signal
transduction is believed to activate, rather than to repress, the
transcription of target genes. Therefore, we hypothesize that Notch signaling
at the four-cell stage represses tbx-37 and tbx-38
transcription through at least one intermediate. The absence of this
intermediate in the ABa blastomere could allow the subsequent,
Notch-independent, expression of TBX-37, TBX-38 in all ABa descendants.
ABa and ABp are born as equivalent cells, and all the differences that
arise between these cells are thought to result from Notch-mediated
interactions. If ABp, in the combined absence of Notch interactions and
TBX-37, TBX-38, can still develop like a wild-type ABp, we would predict that
ABa should be transformed into an ABp-like cell under the same conditions. We
have not tested this prediction directly by constructing a glp-1 tbx-37
tbx-38 triple mutant strain; however, some of our lineage data from
tbx-37 tbx-38 embryos addresses this prediction. The ABa descendant
called ABarp is not signaled by either MS or P2 (see
Fig. 1B), and in a tbx-37
tbx-38 mutant ABarp and its descendants will not contain TBX-37, TBX-38.
Therefore, ABarp in a tbx-37 tbx-38 embryo would be predicted to
develop like the corresponding wild-type ABp descendant, called ABprp. The
ABarp descendants ABarpaa and ABarpap remain on the surface in wild-type
embryos, but ingress into the body cavity in tbx-37 tbx-38 mutant
embryos. Interestingly, the corresponding ABprp descendants (ABprpaa and
ABprpap) ingress into the body cavity in wild-type embryos
(Sulston et al., 1983). The
ABarp descendant ABarppaaa divides into equal sized daughters in wild-type
embryos, but divides asymmetrically to generate a small, posterior daughter
that undergoes programmed cell death in tbx-37 tbx-38 embryos. In
wild-type embryos, the corresponding ABprp descendant (ABprppaaa) divides
asymmetrically to generate a posterior cell death
(Sulston et al., 1983
).
One ABarp descendant in the tbx-37 tbx-38 mutant embryos, called
ABarppp, clearly did not resemble the corresponding ABprp descendant
(ABprppp), and instead appeared nearly wild type
(Table 1). In wild-type
development, ABarppp and its sister are unusual in that they are born from an
anteroposterior cell division and yet have identical fates; these sisters form
bilaterally symmetrical clones of cells on the left and right sides of the
body. All of the other 15 examples of anterior/posterior cell divisions that
occur at the same time in embryogenesis generate sister cells with different
cell fates (Sulston et al.,
1983). Thus, it is possible that the fate of ABarppp is regulated
by unknown, Notch-independent events that are important for bilateral
symmetry.
In summary, our results provide insight into two of the four Notch-mediated interactions that occur in rapid succession in early embryogenesis, and that modify ABa and ABp descendants in distinct ways. We propose that the transcription factors TBX-37, TBX-38 can promote `primary' cell fates independent of Notch. The first Notch-mediated interaction blocks expression of TBX-37, TBX-38 in ABp descendants, thus allowing those cells to adopt novel, `secondary' fates. Next, TBX-37, TBX-38 are expressed in ABa descendants independently of Notch, but shortly after the second Notch interaction. ABa descendants that do not undergo the second Notch interaction assume primary fates, in part through the action of TBX-37, TBX-38. In the ABa descendants that undergo the second Notch-interaction, TBX-37, TBX-38 collaborate with unidentified Notch targets to promote tertiary fates and mesoderm development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alder, M. N., Dames, S., Gaudet, J. and Mango, S. E.
(2003). Gene silencing in Caenorhabditis dlegans by transitive
RNA interference. RNA 9,25
-32.
Austin, J. and Kimble, J. (1987). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell 51,589 -599.[Medline]
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: Cell fate control and signal integration
in development. Science
284,770
-776.
Bowerman, B., Eaton, B. A. and Priess, J. R. (1992). skn-1, a maternally expressed gene required to specify the fate of ventral blastomeres in the early C. elegans embryo. Cell 28,1061 -1075.
Bowerman, B., Draper, B. W., Mello, C. C. and Priess, J. R. (1993). The maternal gene skn-1 encodes a protein that is distributed unequally in early C. elegans embryos. Cell 74,443 -452.[Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. A., Gessler, M., Nemer, M., Seidman, C. E. and Seidman, J. G. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106,709 -721.[CrossRef][Medline]
Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391,695 -697.[CrossRef][Medline]
Chisholm, A. D. and Hodgkin, J. (1989). The mab-9 gene controls the fate of B, the major male-specific blast cell in the tail region of Caenorhabditis elegans. Genes Dev. 3,1413 -1423.[Abstract]
Christensen, S., Kodoyianni, V., Bosenberg., M., Freidman, L.
and Kimble, J. (1996). lag-1, a gene required for
lin-12 and glp-1 signaling in C. elegans is
homologous to human CBF1 and Drosophila Su(H).
Development 122,1373
-1383.
Fitzgerald, K., Wilkinson, H. A. and Greenwald, I.
(1993). glp-1 can substitute for lin-12 in
specifying cell fate decisions in Caenorhabditis elegans.Development 119,1019
-1027.
Fitzgerald, K. and Greenwald, I. (1995).
Interchangeability of Caenorhabditis elegans DSL proteins and
intrinsic signalling activity of their extracellular domains in vivo.
Development 121,4275
-4282.
Gao, D. L. and Kimble, J. (1995). APX-1 can substitute for its homolog LAG-2 to direct cell interactions throughout Caenorhabditis elegans development. Proc. Natl. Acad. Sci. USA 92,9839 -9842.[Abstract]
Garg, V., Kathiriya, I. S., Barnes, R., Schluterman, M. K., King, I. N., Butler, C. A., Rothrock, C. R., Eapen, R. S., Hirayama-Yamada, K., Joo, K., Matsuoka, M., Cohen, J. C. and Srivastava, D. (2003). GATA4 mutations cause human congential heart defects and reveal an interaction with TBX5. Nature 424,443 -447.[CrossRef][Medline]
Greenwald, I., Sternberg, P. W. and Horvitz, H. R. (1983). The lin-12 locus specifies cell fates in Caenorhabditis elegans. Cell 34,435 -444.[Medline]
Hartenstein, V. and Posakony, J. W. (1990). A dual function of the Notch gene in Drosophila sensillum development. Dev. Biol. 142,13 -30.[Medline]
Haun, C., Alexander, J., Stainier, D. Y. and Okkema, P. G.
(1998). Rescue of Caenorhabditis elegans pharyngeal
development by a vertebrate heart specification gene. Proc. Natl.
Acad. Sci. USA 95,5072
-5075.
Hermann, G. J., Leung, B. and Priess, J. R.
(2000). Left-right asymmetry in C. elegans intestine
organogenesis involves a LIN-12/Notch signaling pathway.
Development 127,3429
-3440.
Hiroi, Y., Sudoh, S., Monzen, K., Ikeda, Y., Yazaki, Y., Nagai, R. and Komuro, I. (2001). Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat. Genet. 28,276 -280.[CrossRef][Medline]
Hobert, O. (2002). PCR Fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. BioTechniques 32,728 -730.[Medline]
Horner, M. A., Quintin, S., Domeier, M. E., Kimble, J.,
Labouesse, M. and Mango, S. (1998). pha-4, an HNF-3
homolog, specifies pharyngeal organ identity in Caenorhabditis elegans.Genes Dev. 12,1947
-1952.
Hsueh, Y. P., Wang, T. F., Yang, F. C. and Sheng, M. (2002). Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Nature 404,298 -302.
Hutter, H. and Schnabel, R. (1994).
glp-1 and inductions establishing embryonic axes in C. elegans.Development 120,2051
-2064.
Hutter, H. and Schnabel, R. (1995).
Establishment of left-right asymmmetry in the Caenorhabditis elegans
embryo is a multistep process involving a series of inductive events.
Development 121,3417
-3424.
Iso, T., Hamamori, Y. and Kedes, L. (2003).
Notch signaling in vasclar development. Arterioscler. Thromb. Vasc.
Biol. 23,543
-553.
Kalb, J. M., Lau, K. K., Goszczynski, B., Fukushige, T., Moons,
D., Okkema, P. and McGhee, J. D. (1998). pha-4 is
Ce-fkh-1, a forkhead/HNF-3 alpha, beta, gamma homolog that functions in
organogenesis of the C. elegans pharynx.
Development 125,2171
-2180.
Kaletta, T., Schnabel, H. and Schnabel, R. (1997). Binary specification of the embryonic lineage in Caenorhabditis elegans. Nature 390,294 -298.[CrossRef][Medline]
Kostas, S. A. and Fire, A. (2002). The T-box
factor MLS-1 acts as a molecular switch during specification of nonstriated
muscle in C. elegans. Genes Dev.
16,257
-269.
Lambie, E. J. and Kimble, J. (1991). Two homologous regulatory genes, lin-12 and glp-1, have overlapping functions. Development 112,231 -239.[Abstract]
Lin, R., Thompson, S. and Priess, J. R. (1995). pop-1 encodes an HMG box protein required for the specification of a mesoderm precursor in the early C. elegans embryo. Cell 83,599 -609.[Medline]
Lin, R., Hill, R. J. and Priess, J. R. (1998). POP-1 and anterior-posterior fate decisions in C. elegans embryos. Cell 92,229 -239.[Medline]
Mango, S. E., Thorpe, C. J., Martin, P. R., Chamberlain, S. H.
and Bowerman, B. (1994a). Two maternal genes, apx-1
and pie-1, are required to distinguish the fates of equivalent
blastomeres in the early Caenorhabditis elegans embryo.
Development 120,2305
-2315.
Mango, S., Lambie, E. J. and Kimble, J.
(1994b). The pha-4 gene is required to generate the
pharyngeal primordium of Caenorhabditis elegans.Development 120,3019
-3031.
Mello, C. C., Draper, B. W. and Priess, J. R. (1994). The maternal genes apx-1 and glp-1 and establishment of dorsal-ventral polarity in the early C. elegans embryo. Cell 77,95 -106.[Medline]
Moskowitz, I. P. G., Gendreau, S. B. and Rothman, J. H.
(1994). Combinatorial specification of blastomere identity by
glp-1-dependent cellular interactions in the nematode
Caenorhabditis elegans. Development
120,3325
-3338.
Moskowitz, I. P. G. and Rothman, A. (1996).
lin-12 and glp-1 are required zygotically for early
embryonic cellular interactions and are regulated by maternal GLP-1 signaling
in Caenorhabditis elegans. Development
122,4105
-4117.
Muhlrad, P. J. and Ward, S. (2002).
Spermiogenesis initiation in Caenorhabditis elegans involves a casein
kinase 1 encoded by the spe-6 gene. Genetics
161,143
-155.
Okkema, P. G., Ha, E., Haun, C., Chen, W. and Fire, A.
(1997). The Caenorhabditis elegans NK-2 homeobox gene
ceh-22 activates pharyngeal muscle gene expression in combination
with pha-1 and is required for normal pharyngeal development.
Development 124,3965
-3973.
Newman, A., White, J. G. and Sternberg, P. W.
(1995). The C. elegans lin-12 gene mediates induction of
ventral uterine specialization by the anchor cell.
Development 121,263
-271.
Page, B. D., Zhang, W., Steward, K., Blumenthal, T. and Priess, J. R. (1997). ELT-1, a GATA-like transcription factor is required for epidermal cell fates in Caenorhabditis elegans embryos. Genes Dev. 11,1651 -1661.[Abstract]
Papaioannou, V. E. and Silver, L. M. (1998). The T-box gene family. BioEssays 20, 9-19.[CrossRef][Medline]
Parks, A. and Muskavitch, M. A. T. (1993). Delta function is required for bristle organ determination and morphogenesis in Drosophila. Dev. Biol. 157,484 -496.[CrossRef][Medline]
Park, F. D. and Priess, J. R. (2003).
Establishment of POP-1 asymmetry in early C. elegans embryos.
Development 130,3547
-3556.
Priess, J. R., Schnabel, H. and Schnabel, R. (1987). The glp-1 locus and cellular interactions in early C. elegans embryos. Cell 51,601 -611.[Medline]
Ruvkun, G. and Hobert, O. (1998). The taxonomy
of developmental control in Caenorhabditis elegans.Science 282,2033
-2041.
Shelton, C. A. and Bowerman, B. (1996).
Time-dependent responses to glp-1-mediated inductions in early C.
elegans embryos. Development
122,2043
-2050.
Schnabel, R. and Priess, J. R. (1997). Specification of cell fates in the early embryo. In: C. elegans II, pp. 361-383. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100,64 -119.[Medline]
Wayner, E. A. and Carter, W. G. (1987). Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J. Cell Biol. 105,1873 -1884.[Abstract]
White, P. H., Farkas, D. R., McFadden, E. E. and Chapman, D.
L. (2003). Defective somite patterning in mouse embryos with
reduced levels of Tbx6. Development
130,1681
-1690.
Woollard, A. and Hodgkin, J. (2000). The
Caenorhabditis elegans fate-determining gene mab-9 encodes a
T-box protein required to pattern the posterior hindgut. Genes
Dev. 14,596
-603.
Related articles in Development: