Essential role for Notch
signaling in restricting
Nareg J.-V. Djabrayan, Nathaniel R. Dudley,
Erica M. Sommermann, and Joel H. Rothman1
Department of Molecular, Cellular, and Developmental Biology,
Neuroscience Research Institute, University of California
at Santa Barbara, Santa Barbara, California 93106, USA
We report that Notch signaling is essential for the switch
from developmental plasticity to commitment during
Caenorhabditis elegans embryogenesis. The GLP-1 and
LIN-12 Notch receptors act to set a memory state that
affects commitment of cells arising from the major ec-
todermal progenitor (AB blastomere) several cell divisions
later, thereby preventing their forced reprogramming by
an endoderm-determining transcription factor. In con-
trast to Notch-dependent cell fate induction, this activity
is autonomous to the AB lineage, is independent of the
known cell fate-inducing Notch ligands, and requires a
putative secreted Notch ligand, Delta Serrate Lag-3 (DSL-3).
Thus, Notch signaling promotes developmental commit-
ment by a mechanism that is distinct from that involved in
specifying cell fates.
Supplemental material is available for this article.
Received June 27, 2012; revised version accepted September
Fundamental to our understanding of developmental and
stem cell biology is how cells switch from a pluripotent to
a developmentally committed state. Methods for growing
and engineering tissues and organs in vitro depend criti-
cally on the ability to manipulate this process. All somatic
cells in early Caenorhabditis elegans embryos have been
shown to be pluripotent, as evidenced by their capacity to
be reprogrammed into cells of all three germ layers in
response to forced expression of cell fate-regulating tran-
scription factors (Horner et al. 1998; Zhu et al. 1998;
Gilleard and McGhee 2001; Fukushige and Krause 2005).
Later in embryogenesis, at about the time that the
endoderm progenitor, or E cell, has progressed beyond
three rounds of division (the 8E stage) (Joshi et al. 2010),
cells become refractory to reprogramming by these factors,
marking a major transition from plasticity to restric-
tion in developmental potential (e.g., as can be observed
by challenging normally nonendodermal precursor cells
to undergo endoderm development) (Supplemental Fig.
S1A). While cell-autonomous mechanisms in this critical
developmental transition (e.g., Joshi et al. 2010) are known,
the action of cell-extrinsic signaling in this process has not
been well characterized.
The contact-dependent signal transduction mechanism
known as Notch signaling is broadly deployed to direct
Tsakonas and Muskavitch 2010). In C. elegans, Notch
signaling is known primarily from its role in specifying
cell identities and regulating cell behavior in the embryo,
larvae, and adult germline (Kodoyianni et al. 1992; Berry
et al. 1997; Chen and Greenwald 2004; Shaye and
Greenwald 2005; McGovern et al. 2009). Here we show
that Notch pathway components are essential for a com-
pletely distinct function independent of their action in
specifying cellular differentiation: regulating the ability
of embryonic cells to be reprogrammed. We show that the
Notch receptor GLP-1 restricts the lineage of AB, the
major embryonic ectoblast, from being reprogrammed
into endoderm by a mechanism that is autonomous to
the AB lineage and therefore distinct from the known
early embryonic inductions. Furthermore, we implicate
a presumptive Notch ligand, Delta Serrate Lag-3 (DSL-3),
in the process that prevents cells in the AB lineage from
becoming reprogrammed but not in specifying cell iden-
tity in this lineage per se. These findings reveal that Notch
controls the transition from plasticity to committed differ-
entiation independent of its previously known action in
cell fate specification.
Results and Discussion
Contact-dependent signaling through Notch-type recep-
tors is recursively and widely deployed to specify the
identity of many descendants of AB, the anterior blasto-
mere of the two-cell C. elegans embryo, which generates
most of the ectoderm (Priess et al. 1987; Moskowitz and
Rothman 1996; Schnabel and Priess 1997; Priess 2005). As
Notch signaling functions most prominently prior to
the plasticity / commitment transition, we wondered
whether it might also act in restricting developmental
plasticity. Indeed, we found that eliminating the function
of the GLP-1 Notch receptor greatly extends the period
during which nonendodermal cells can be reprogrammed
into endoderm in response to the endoderm-promoting
END-3 GATA transcription factor (Fig. 1A). In the ab-
sence of GLP-1 function, lineage reprogramming, evident
by expression of several endodermal markers, can occur
as late as the 20E stage, well after developmental plastic-
ity is normally lost in wild-type embryos (Fig. 1B,C;
Supplemental Fig. S3). This period of extended plasticity
does not continue indefinitely, however, as cells do not
respond to ectopic END-3 when its expression is induced
late in morphogenesis (i.e., at approximately the ‘‘twofold
stage’’) (Fig. 1A). This ability of late embryos to undergo
ectopic endoderm development is not the result of a
general defect in differentiation, as glp-1(?) embryos
undergo timely development and differentiation (Hutter
and Schnabel 1994; Moskowitz et al. 1994; Schnabel and
Priess 1997; data not shown), albeit with altered cell
lineage patterns and fates, owing to misspecification of
cells within the AB lineage. GLP-1 appears to act at least
in part through canonical Notch signaling in this process:
We found that the LAG-1 transcription factor, which
transduces Notch inductive signals (Christensen et al.
1996), is also required for the temporal restriction in
developmental plasticity (Table 1).
[Keywords: Notch; reprogramming; embryo culture; commitment; plasticity]
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.199588.112.
2386GENES & DEVELOPMENT 26:2386–2391 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org
We found that when GLP-1 is inactivated prior to the
28-cell (2E) stage, embryos remain competent to respond
to late (up to the 20E stage) ectopic expression of END-3
(Fig. 1A,D); reactivating GLP-1 function after the 28-cell
stage does not reverse this effect (Fig. 1D). In contrast,
when GLP-1 is inactivated only after the 28-cell stage,
embryos become developmentally committed at the nor-
mal time (approximately 8E stage) (Fig. 1D). These results
suggest that GLP-1/Notch sets up a memory state in the
early embryo that activates the plasticity / commitment
transition later in embryogenesis.
After the early inductive interactions in the AB lineage,
mediated by maternal GLP-1, zygotically expressed GLP-
1 and its paralog, LIN-12, act redundantly to induce the
fates of a small subset of AB-derived cells (Hutter and
Schnabel 1994; Moskowitz and Rothman 1996; Henderson
et al. 1997). Thus, LIN-12 might compensate for the
absence of GLP-1 in regulating developmental plasticity
after the 28-cell stage. We found that while embryos
lacking only LIN-12 function become developmentally
committed at the same time (approximately 8E stage) as
in wild type, embryos lacking both GLP-1 and LIN-12
after the 28-cell stage can be provoked to produce ectopic
endoderm late in development (Fig. 1D). Consistent with
the known requirement of early GLP-1 activity for LIN-12
expression (Moskowitz and Rothman 1996), we observed
no significant difference in the ability of embryos to be
developmentally reprogrammed when GLP-1 activity
was absent throughout embryogenesis in lin-12(RNAi)
embryos compared with lin-12(+), embryos (Fig. 1D). It is
possible that Notch signaling is necessary only to the 8E
stage to restrict plasticity. Indeed, we found that lin-12(?)
embryos in which GLP-1 activity was maintained until
the 8E stage and then subsequently inactivated were
resistant to END-3-mediated reprogramming (Fig. 1D).
These results define an apparently continuous require-
ment for Notch function in the transition from a plastic
to a committed state, including an essential early (<28-cell)
requirement mediated by GLP-1 alone and a later (>28-cell)
requirement in which either GLP-1 or LIN-12 appears to
As with the known early Notch-mediated embryonic
inductions, we found that GLP-1/Notch function in
restricting developmental plasticity is limited to AB
descendants. While partial wild-type embryos derived by
laser ablation of either AB or its sister, P1, showed the
restriction of developmental plasticity
Test of Notch signaling components in the
RNAi Embryos with ectopic elt-2Tgfp
1.44% 6 1.35% (n = 140)
0% (n = 43)
1.54% 6 1.39% (n = 65)
0% (n = 40)
25.8% 6 5.6% (n = 62)a
0% (n = 32)
Percent of embryos showing widespread elt-2TGFP expression
after END-3 induction at the 20E stage is reported. L4440 is the
control feeder strain.
aFisher’s exact test, P < 0.01, compared with control L4440 strain.
Proportion of terminal embryos with ectopic elt-2TGFP in wild-type (WT) and glp-1(e2144) embryos following induction of END-3 at the
indicated stage. ‘‘Early 20E’’ indicates that END-3 was induced in embryos shortly after the last E descendants were born (approximately ‘‘bean’’
to ‘‘comma’’ stage). ‘‘Late 20E’’ indicates that END-3 was induced at approximately the ‘‘twofold’’ stage of morphogenesis, based on time from
the two- to four-cell stage. Numbers in bars indicate total embryos carrying the hs-end-3 array scored. (B) Proportion of terminal embryos with
ectopic expression of the late gut differentiation marker IFB-2, detected with antibody MH33 (Zhu et al. 1998) following induction of END-3 at
the 20E stage. (C) Expression of intestinal differentiation markers following late activation of END-3 in embryos of the indicated genotype. (D)
Timelines of temperature shift experiments. Embryos were held at the indicated temperature [permissive (15°C) or nonpermissive (25°C) for the
glp-1(e2144) mutation] and shifted at the indicated developmental stage. (Top) The known temperature-sensitive period for GLP-1 lethality
(Priess et al. 1987) and the normal period of developmental plasticity (‘‘competency window’’) are shown on the developmental time line. lin-
12(?) indicates that the gene was knocked down by RNAi in the mothers of the embryos where indicated. Pie diagrams show the proportion of
embryos with ectopic endoderm; the total number of embryos carrying the END-3 transgene is indicated below each. In B–D, hs-end-3 was
activated at a stage equivalent to the 20E stage in wild-type embryos. Error bars represent standard error. (*) Fisher’s exact test, P < 0.01.
Extended period of developmental plasticity in embryos lacking GLP-1/Notch function, and temporal requirement for GLP-1. (A)
Notch signaling restricts plasticity
GENES & DEVELOPMENT2387
same temporal restriction to developmental reprogram-
ming as in intact embryos (Supplemental Fig. S4A,C), AB-
derived partial embryos lacking GLP-1 could be provoked
to develop endoderm well after the period in which
developmental plasticity is normally lost (Fig. 2B,D). In
contrast, this temporal extension of plasticity was not
seen in P1-derived glp-1(?) partial embryos (Supplemen-
tal Fig. S4B), demonstrating the AB specificity.
Given these observations and the known role of P1
descendants as the source of early inductive Notch
signals that pattern the AB lineage (summarized in Fig.
2A; Priess et al. 1987; Schnabel and Priess 1997; Priess
2005), it was reasonable to suppose that the signals
required to restrict developmental plasticity in AB de-
scendants later in development might also arise from P1
descendants (Model A, Fig. 2A). However, several obser-
vations indicated that this is not the case. First, although
the entire AB lineage appears to be resistant to de-
velopmental reprogramming after the 8E stage in wild-
type embryos, two of the AB great-granddaughters, while
expressing the GLP-1/Notch receptor, never receive Notch
signals and would therefore beexpected to show extended
developmental plasticity (Hutter and Schnabel 1994;
Moskowitz et al. 1994). In addition, we found that APX-1,
the Notch ligand that induces the fate of half of the AB-
derived cells (Mango et al. 1994; Mello et al. 1994), is not
required to restrict developmental plasticity (Table 1).
Furthermore, cell ablations (Fig. 2B,D; Supplemental Fig.
S4A) indicated that a viable P1cell is not required for
commitment of AB descendants to a nonendodermal fate.
However, this experiment does not eliminate the
caveat that P1-derived signals might be produced
by the ablated cell (e.g., Goldstein 1992). To
eliminate all potential P1-derived Notch signal,
we physically isolated and cultured AB blasto-
meres (Edgar and Goldstein 2012) and tested for
GLP-1-dependent restriction of developmental
plasticity. This manipulation is known to prevent
all Notch signaling to the AB lineage (Gendreau
et al. 1994; Moskowitz et al. 1994). The resultant
partial embryos differentiated (Supplemental Fig.
S5A,B) and produced endoderm when induced to
express END-3 at early stages (Supplemental Fig.
S5C,D). However, although they do not experience
Notch signal from P1descendants, the GLP-1-de-
pendent block to reprogramming nonetheless per-
sisted in such AB-derived embryos. Specifically,
endoderm differentiation, as evident by elt-2TGFP
expression as well as the presence of birefringent
gut granules, an endogenous marker of endoderm
differentiation (Babu 1974), could be activated in
late glp-1(?) but not glp-1(+) AB-derived partial
embryos (Fig. 2C,E; data not shown). Thus, poste-
rior-derived Notch signals are not necessary for
developmental commitment; rather, the plasticity-
regulating signals apparently arise from within
the AB lineage (Model B, Fig. 2A). These intra-AB
signals cannot be attributed to the few known
(and very limited) intra-AB inductions received
by LIN-12, as LIN-12 is not expressed in embryos
lacking P1-derived Notch signals (Moskowitz
and Rothman 1996).
The only Notch ligand known to be expressed
and functioninthe embryonic ABlineage is LAG-2,
which activates zygotic expression of lin-12 and
glp-1 (Moskowitz and Rothman 1996). However,
we found that lag-2 knockdown did not affect
the ability of late embryos to be reprogrammed
(Table 1), suggesting the requirement for an un-
identified AB-specific Notch ligand. The C. elegans
genome encodes several other DSL-like Notch
ligands (Chen and Greenwald 2004). We found
that knocking down either of two DSL-encoding
genes, dsl-1 or dsl-3, extended the period of de-
velopmental plasticity (Fig. 3A,B). Of these, only
dsl-3 showed a requirement for commitment in
the AB lineage specifically when partial em-
bryos, obtained by laser ablation of AB or P1,
were analyzed (Fig. 3C,D). Thus, like GLP-1/
Notch, DSL-3, which is predicted to be a secreted
AB lineage. (A, left panel). Summary of early known cell fate-inducing Notch
signals activated by P1descendants. Gray-filled cells, derived from P1, signal their
AB-derived Notch-expressing neighbors. Black outlines indicate cells expressing
the GLP-1/Notch receptor. Cells in which Notch signal transduction has been
received by signaling from P1descendants contain black nuclei. (Right panel)
Alternative models for Notch-dependent regulation of developmental plasticity.
Circles at the top of each model represent isolated AB cells with (heavy lines) or
without (light lines) GLP-1 present. Model A: Inductive signals from P1 de-
scendants both activate cell fates and restrict developmental plasticity. Model B:
AB-derived signals, distinct from the cell fate-inducing signals arising from P1
descendants, restrict developmental plasticity. (B) Proportion of P1-ablated
embryos from wild-type and glp-1(e2144) embryos that express ectopic elt-2T
GFP following late induction of END-3 expression. (C) Proportion of partial
embryos obtained from in vitro culturing of physically isolated AB blastomeres
from wild-type and glp-1(e2144) embryos that express ectopic elt-2TGFP. (D) elt-2T
GFP expression in wild-type and glp-1(e2144) embryos in which AB was isolated
by laser ablation followed by late induction of END-3 expression. Ablated
blastomeres are denoted by dashed lines. (E) elt-2TGFP in partial embryos
obtained from physically isolated AB blastomeres. In all experiments, END-3
was induced by heat shock when the number of cells corresponded approxi-
mately to the 20E stage in intact wild-type embryos. In B and C, numbers in
the bars represent the total number of embryos carrying the END-3 transgene.
(*) Fisher’s exact test, P = 0.01; (**) Fisher’s exact test, P < 0.01. Error bars
represent standard error.
Notch signals regulating developmental plasticity are specific to the
Djabrayan et al.
2388GENES & DEVELOPMENT
protein (Chen and Greenwald 2004), apparently acts
specifically on AB descendants to restrict developmental
Consistent with its potential action on Notch signal-
ing to the AB lineage, microarrays (Baugh et al. 2003)
and expression patterns reported in the NEXTDB data-
base (The Nematode Expression Pattern Database,
http://nematode.lab.nig.ac.jp) indicate that dsl-3 ex-
pression overlaps temporally with that of the GLP-1
and LIN-12 Notch receptors. Moreover, we detected dsl-3
transcripts by in situ hybridization throughout the em-
bryo as early as the two-cell stage, with stronger, more
restricted staining primarily in AB-derived embryonic
cells later in development (Supplemental Fig. S6). While
knockdown of dsl-3 under conditions that eliminate a
detectable message (Supplemental Fig. S6) impairs de-
velopmental commitment, we observed no overt pheno-
type of larvae arising from such embryos, consistent with
other reports (Kamath et al. 2003; Sonnichsen et al. 2005).
Thus, while dsl-3 transcripts are present throughout the
early embryo, DSL-3 may block reprogramming by acting
specifically through Notch receptors expressed only in
AB descendants. These findings suggest two fundamen-
tally different modes of action for Notch signaling in
development, with one set of ligands that controls spec-
ification of cell identity and another that mediates an
auxiliary role in regulating developmental plasticity (Sup-
plemental Fig. S7).
Previous work identified a role for a mem-
ber of the C. elegans polycomb repressor
complex, MES-2, in restricting developmen-
tal plasticity (Yuzyuk et al. 2009). In that
study, it was shown that removal of mes-2
function enhances developmental plastic-
ity at the 8E stage. It is therefore possible
that MES-2 acts downstream from Notch
signaling to promote commitment to dif-
ferentiation. We found that RNAi knock-
down of mes-2 in glp-1(?) embryos did not
significantly (P = 1) increase the fraction of
embryos (53.9% 6 9.8%; n = 26) that were
reprogrammed, consistent with the possibil-
ity that Notch signaling and MES-2 function
in the same pathway to restrict develop-
mental plasticity. However, the findings
that MES-2 function is not lineage-specific
(Yuzyuk et al. 2009) and that inactivation of
GLP-1 causes cells to remain plastic longer
than those in embryos lacking MES-2 func-
tion suggest that MES-2 may act in a global
pathway with multiple inputs, while Notch
apparently acts in an AB-specific pathway
that may impinge on MES-2 and other regu-
lators of chromatin function.
In other systems, Notch signaling acts to
coordinate proliferation and differentiation,
as observed, for example, with mammalian
Notch1, which is necessary for both the
proliferation of neuronal stem cell popula-
tions and their differentiation into specific
neuronal types (Hitoshi et al. 2002; Wang
et al. 2009; Ables et al. 2010; Zhou et al.
2010; Matsumoto et al. 2011). We showed
that in C. elegans, Notch can act not only
as a factor in specifying cell types, but also,
through an apparently distinct process, in the general
restriction of developmental plasticity. The diversifica-
tion of Notch function through the action of different
ligands may point to a new role for Notch signaling in
regulating the multipotential, stem cell-like properties of
Materials and methods
Worm culture and strains
Unless otherwise noted, strains were reared at 20°C on NGM agar plates
fed on OP50 Escherichia coli. glp-1(e2144) worms were reared at 15°C,
and all experiments with these mutants were performed at 25°C, except
where indicated. The following strains were constructed for this study:
JR1753 unc-119(ed4) III; wEx690 [unc-119(+) hsp16-2/41Tend-3(+)];
wIs84 [rol-6(su1006) elt-2TGFP] and JR3279 unc-119(ed4) glp-1(e2144ts)
III; wEx690 [unc-119(+) hsp16-2/41Tend-3(+)]; wIs84 [rol-6(su1006) elt-2T
GFP]. Strain JG7 cals6 [hsp16-2Telt-1(+)]; ijIs12 [dpy7TGFP rol-6(su1006)]
was obtained from John Gilleard.
E. coli RNAi feeding strains for glp-1, lin-12, lag-2, dsl-1, dsl-4, lag-1, and
ref-1 were obtained from the Ahringer RNAi library (Fraser et al. 2000;
Kamath et al. 2003). The dsl-2, dsl-3, dsl-5, dsl-7, and apx-1 genes were
cloned into plasmid L4440 using appropriate primers carrying added
restriction sites to facilitate cloning (sequences furnished on request)
and were transformed into HT115 for RNAi feeding. In the case of glp-1,
apx-1, and lag-1, which show lethal RNAi phenotypes, the efficacy of
(A) Proportion of embryos subjected to RNAi of the indicated gene that showed ectopic
elt-2TGFP expression. (B) Endogenous and ectopic elt-2TGFP in terminal dsl-1(RNAi)
and dsl-3(RNAi) embryos. (C) elt-2TGFP in terminal P1-ablated wild-type and dsl-3(RNAi)
embryos. Ablated blastomeres are denoted by dashed lines. (D) Proportion of isolated P1
or AB descendants of wild-type and dsl-3(RNAi) embryos expressing ectopic elt-2TGFP.
In all experiments, END-3 was induced by heat shock when the number of cells
corresponded approximately to the 20E stage in intact wild-type embryos. Numbers in
bars are the total number of embryos carrying the END-3 transgene. (*) Fisher’s exact
test, P < 0.01. Error bars represent standard error.
dsl-3 is required for commitment to differentiation in the AB lineage.
Notch signaling restricts plasticity
GENES & DEVELOPMENT2389
RNAi was determined by scoring for embryonic lethality. L3 worms
were fed on RNAi feeding bacteria for 3 d at 15°C. Embryos were
dissected from gravid adults on the third day and used in heat-shock
Analysis of responsiveness to cell fate reprogramming
Embryos from strain JR1753 or the indicated mutant or knockdown
strains were isolated at the two-cell stage, and END-3 expression was
induced via heat shock after they had been allowed to develop to various
stages of E development. Embryos were returned to 20°C, allowed to
develop overnight, and scored for expression of elt-2TGFP. We found that
induction of END-3 at any stage leads to ectopic expression of elt-2TGFP
in a low number of small nuclei; however, this expression did not appear
to reflect bona fide reprogramming, as ectopic expression of other markers
of gut differentiation was not observed in such cells. We scored only
embryos with widespread elt-2TGFP expression as positive in all exper-
iments (e.g., Supplemental Fig. S2). For analysis of differentiation markers,
terminal embryos were prepared for immunofluorescence. In all heat-
shock experiments, embryos were shifted for 30 min to 33°C, with the
exception that embryos from the laser ablation and in vitro culture
experiments were heat-shocked for 15 min at 33°C.
Temperature shift experiments
To block GLP-1 function continuously, glp-1(e2144) embryos were
isolated at the two- to four-cell stage and incubated immediately at the
nonpermissive temperature (25°C) through to the 20E stage, at which
point they were heat-shocked. To block GLP-1 function later, glp-1(e2144)
embryos were isolated at the permissive temperature (15°C) in a 15°C
temperature-controlled room. Following incubation at 15°C past the
28-cell stage or 8E stage, embryos were shifted to the nonpermissive
temperature and incubated until the 20E stage, at which point they were
heat-shocked. Shift-down assays were performed by isolating and in-
cubating glp-1(e2144) embryos at the nonpermissive temperature from
the two- to four-cell stages and shifting to the permissive temperature
after the 28-cell stage. After allowing them to develop to the 20E cell
stage, embryos were heat-shocked.
AB or P1blastomeres were ablated at the two-cell stage with a pulsed laser
microbeam (Photonic Instruments, Inc.) as described in Bowerman et al.
(1992). After ablation, embryos from either wild-type or glp-1(e2144)
worms were incubated to the equivalent of the 20E stage at 25°C, based
on observation of total cell number. Embryos were then heat-shocked for
15 min at 33°C and incubated overnight at 20°C before scoring.
In vitro culture
The methods for culturing isolated blastomeres were adapted from Edgar
and Goldstein (2012). After enzymatic digestion of the eggshell, blasto-
meres were separated by forcing embryos through a pulled glass needle.
AB or P1blastomeres were identified by size and mounted in 14 mL of
Edgar’s growth medium on a glass slide under a coverslip mounted with
clay feet. Slides were sealed with Vaseline to prevent desiccation. To
assess the ability of cells grown in isolation to differentiate, AB blasto-
meres from strain JG7 were incubated overnight at 20°C and scored for
expression of the epidermal marker dpy-7TGFP (Gilleard and McGhee
2001). P1blastomeres from strain JR3339 were grown overnight at 20°C
and scored for expression of muscle marker unc-54TGFP. To test whether
cultured AB blastomeres exhibit the same transition from developmental
plasticity to commitment as intact embryos, AB isolates from wild-type
embryos were allowed to develop at 20°C to the equivalent of the 2E and
20E stage, as determined by cell number; heat-shocked for 15 min at 33°C;
returned to 20°C; and scored for elt-2TGFP expression after overnight
incubation. To test for GLP-1 dependence in commitment to differenti-
ation, AB blastomeres were isolated from wild-type and glp-1(e2144)
embryos, grown in vitro at 25°C to the equivalent of the 20E stage, and
heat-shocked for 15 min at 33°C. After overnight incubation at 20°C,
descendants were scored for elt-2Tgfp expression.
In situ hybridization
Mixed-stage embryos from N2, dsl-1(ok810), dsl-3(RNAi), or glp-1(e2144)
worms were stained with DIG-labeled RNA probes for dsl-1 and dsl-3.
Probes were made as described (http://www.faculty.ucr.edu/mmaduro)
with appropriate primers (sequences provided on request). BLASTN
analysis of the genomic sequences amplified by these primers indicated
that only the targeted gene would be predicted to hybridize significantly.
Embryos obtained from bleached gravid adults were forced through
27.5-gauge needles to break up adult corpses and then washed three times
in M9. Embryos in M9 suspension were immediately dispensed in 10-mL
aliquots onto 14 3 14-mm square wells of polylysine-coated slides, covered
with coverslips, and frozen. After freeze-crack, the Kohara Laboratory
protocol for ‘‘large-scale fixation of embryos’’ was followed (http://nematode.
lab.nig.ac.jp/method/protocol.php?docbase=insitu_embryo), replacingthe de-
hydration (C.II.11–C.II.1112) and proteinase K treatment steps (D.I.1–D.I.19)
with two 5-min washes in PBT. Hybridizations and subsequent warm
temperature washes were performed at 58°C. For probe detection, slides
were immersed in 400 mL of premixed NBT+BCIP solution (Roche
Diagnostics GmbH, reference 11-681-451-001) mixed with 40 mL of
staining buffer and developed overnight at room temperature.
Imaging and immunofluorescence analysis
The embryonic stage in any given experiment was determined by
observing the number of elt-2Tgfp-expressing cells with an Olympus
SZX12 fluorescence dissecting microscope. Terminal embryos were
scored for ectopic elt-2Tgfp expression on either a Zeiss Axioskop 2 or
Nikon Microphot SA fluorescence compound microscope. Embryos
were mounted on 3% agar pads in egg salts (Edgar and Goldstein 2012).
Imageswereprocessedwith NIHImageJ.For immunofluorescenceanalyses,
embryos were mounted and stained with antibodies MH33 and 1CB4, as
described in Zhu et al. (1998).
We are grateful to M. Kourakis, B. Birsoy, L. Chen, P.M. Joshi, and
J. Casanova for comments on the manuscript. Some nematode strains
were provided by the Caenorhabditis Genetics Center, which is funded by
the NIH National Center for Research Resources (NCRR). N.J.-V.D. was
supported in part by a training grant from the California Institute of
Regenerative Medicine. This work was supported by grants from the NIH
(HD062922) and March of Dimes (FY2007-804) to J.H.R.
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