Department of Molecular, Cellular and Developmental Biology, University
of Michigan, Ann Arbor, MI 48109, USA
* Present address: Department of Molecular Biology, UT Southwestern Medical
Center, 6000 Harry Hines Boulevard, NA8.510, Dallas, TX 75390-9148, USA
Author for correspondence (e-mail:
rolf{at}umich.edu)
Accepted 14 March 2003
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SUMMARY |
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Key words: Drosophila, Heart, Cell cycle regulators, Asymmetric cell division, numb, Notch, CycA, Rca1, dacapo, fizzy-related
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INTRODUCTION |
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The Drosophila heart originates from bilaterally symmetrical rows
of precursor cells in the dorsal mesoderm, which express the homeobox gene
tinman (Bodmer, 1993;
Azpiazu and Frasch, 1993
). As a
consequence of inductive signals from the adjacent ectoderm, tinman
expression is confined to the dorsalmost region of the mesoderm, defined as
the cardiac mesoderm (Frasch,
1995
; Wu et al.,
1995
; Lockwood and Bodmer,
2002
). Within this competence domain, cardiac progenitors emerge
as cell clusters that express a distinct combination of transcription factors,
including even-skipped (eve), ladybird
(lb), seven-up (svp), odd-skipped
(odd) and tinman itself, which are thought to be involved in
conferring the appropriate differentiation pathway to these clusters
(Ward and Skeath, 2000
;
Jagla et al., 2002
;
Han et al., 2002
). The
initiation of these cell clusters of different developing fates is probably
mediated by extrinsic, inductive mechanism, which is determined by their
position in the cardiogenic domain. Subsequently, a defined pattern of
lineages generates the final diversity of cell types
(Ward and Skeath, 2000
). Some
cardiac progenitors divide symmetrically, whereas others undergo stereotyped
asymmetric cell divisions.
Two types of asymmetric cell division in the cardiac mesoderm have been
described: the progenitors of the Svp lineage divide asymmetrically to
generate Svp myocardial cells (SMC) and Svp-Odd pericardial cells (SOPC)
(Ward and Skeath, 2000); the
progenitors of the mesodermal Eve lineage generate Eve pericardial cells (EPC)
and muscle founders DA1 and DO2 (Park et
al., 1998
; Halfon et al.,
2000
; Carmena et al.,
2002
; Han et al.,
2002
). These two types of asymmetric cell division are similar in
the sense that they both generate myogenic versus non-myogenic sibling cell
fates. The myocardial and muscle founder cells have many characteristics of
muscle identity in common, which is excluded in pericardial cell
differentiation. For example, they both express the muscle differentiation
gene Mef2 (Bour et al.,
1995
; Lilly et al.,
1995
). Previous studies suggest that the Notch pathway plays an
important role in determining these alternative cell fate decisions
(Park et al., 1998
;
Carmena et al., 1998
;
Carmena et al., 2002
;
Ward and Skeath, 2000
). In
mutants of the Notch antagonist encoded by numb (reviewed by
Jan and Jan, 1998
), the number
of pericardial SOPC and EPC is increased, accompanied by a loss of myocardial
SMC and of DA1 muscles. Conversely, when numb is overexpressed in the
mesoderm, EPCs are not formed, only DA1 muscles. Moreover, when a
constitutively active form of Notch, Notch intracellular domain or N(icd), is
expressed in the mesoderm, the mesodermal Eve lineage is almost completely
eliminated, suggesting that Notch activity inhibits the formation of
progenitors at an early stage. However, when a temperature-sensitive allele of
Notch is used to eliminate Notch function at the time when
the Eve progenitors divide, EPCs but not DA1 muscle fail to form
(Park et al., 1998
). Thus,
Notch seems to have a dual function, as it is required both for
progenitor specification and for asymmetric cell fate determination of the
same lineage. It also appears that the Notch pathway is required for
specifying pericardial as opposed to myocardial or muscle founder cell fate in
both the Svp and the Eve lineages. It is not known, however, if the
Notch-dependent cell fate decision is made after cell division in the progeny
cells or if it is already initiated in the progenitors before division.
Studying these Notch-mediated asymmetric cell divisions in the
context of the cell cycle may provide insights into the coordination of cell
fate and cell division. Thus, preventing cell division of asymmetric cell
divisions allows us to investigate whether precursors blocked in cell cycle
progression are predetermined or biased in their cell fate decision, or
whether this is a necessary prerequisite for alternative cell fate
determination. Moreover, blocking the mesodermal cell division at
progressively later stages will enable us to get a better understanding of the
cell lineages in the heart. Previous studies have followed marker gene
expression and used the Flp-FRT-based lineage tracing method to address this
question (Park et al., 1998;
Ward and Skeath, 2000
;
Carmena et al., 1998
;
Carmena et al., 2002
), but
some of the details of these lineages are still unclear.
Several genes have been shown to arrest cell divisions at different stages
and cell cycle number within the ectoderm. It is believed that most cells in
Drosophila undergo three rounds of cell division after blastoderm
that are partially synchronous; e.g. slightly earlier in the mesoderm than in
the ectoderm (Foe, 1989;
Campos-Ortega and Hartenstein,
1997
). Later, specialized embryonic tissues, such as the nervous
system, undergo further cell divisions
(Bodmer et al., 1989
;
Foe, 1989
;
Lu et al., 2000
). The last
round of global cell division, mitosis 16, is blocked in CyclinA
(CycA) or Rca1 mutants
(Knoblich and Lehner, 1993
;
Dong et al., 1997
). By
contrast, mitosis 16 is not obviously affected by CyclinB
(CycB) mutants. However, double mutants of CycA and
CycB act synergistically and arrest cell division of most ectodermal
cells at the G2/M transition of cycle 15
(Knoblich and Lehner, 1993
).
In string (stg, cdc25 in yeast) mutant embryos, mesodermal
cells fail to enter metaphase of mitosis 14
(Foe, 1989
;
Edgar and O'Farrell, 1989
;
Edgar and O'Farrell, 1990
). In
addition to these genes that are required for cell cycle progression, some
other cell cycle genes have been shown to be required for cells to exit the
cell cycle. These include dacapo (dap)
(Lane et al., 1996
) and
fizzy related (fzr; rap FlyBase)
(Sigrist and Lehner, 1997
).
dap encodes a CDK inhibitor that is necessary for exiting the cell
cycle at the appropriate time, whereas fzr negatively regulates the
levels of cyclins A, B and B3, and is required for cyclin removal during G1
(when cell proliferation stops). Loss of either gene causes cell division
progression through an extra cycle. Conversely, premature overexpression of
dap or fzr in transgenic embryos inhibits mitosis and
results in cell division arrest (Lane et
al., 1996
; Sigrist and Lehner,
1997
).
In this study, we examined the formation of cardiac cell types in cell cycle mutants and in embryos in which cell cycle inhibitors are overexpressed. We found that the cardiac progenitors continue to differentiate in the absence of cell division. Interestingly, the progenitors of the asymmetric Eve and Svp lineages always adopt a myogenic cell fate, presumably because Notch activation is prevented in the progenitor because of the presence of Numb. To test this, we arrested cell division in numb mutants or overexpressed N(icd) in these progenitors, and found that they adopt a non-myogenic pericardial cell fate. These data indicate that genes normally involved in the cell fate decisions during asymmetric cell division also determine the progenitor cell fate. By contrast, cell fate decisions with symmetric cardiac lineages are not influenced by the presence or absence of Notch activity. Our data suggest that cell cycle genes act in concert with the Notch pathway to generate the diversity of cell types in the Drosophila heart, and that the bHLH transcription factor Suppressor-of-Hairless [Su(H)] mediates this activity. We speculate that an increase in cell type diversity can be achieved by adopting the Numb/Notch system to generate asymmetry within any lineage of an organism.
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MATERIALS AND METHODS |
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Immunohistochemistry and microscopy
Embryos from different lines were collected and stained with various
antibodies as previously described (Han et
al., 2002). The following primary antibodies were used:
anti-ß-galactosidase 1:300 (Promega); anti-Eve 1:10,000
(Frasch et al., 1987
);
anti-Tin 1:500 (Venkatesh et al.,
2000
); anti-Mef2 1:1000 (Lilly
et al., 1995
); and anti-Lbe 1:40
(Jagla et al., 1997
). FITC- or
Cy3-conjugated secondary antibodies (from Jackson Laboratories) were used to
recognize the primary antibodies. Images were obtained with a Zeiss LSM510
confocal microscope.
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RESULTS |
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Distinct lineages of the tinman-expressing myocardial cells
in the anterior two segments of the heart
By comparing the Mef2 and tinman expression in both
wild-type and cell cycle mutants, we noticed that the pattern of cardiac cell
types is different in the anterior two segments (T3 and A1). The embryonic
heart is formed approximately within segment T3 to A8, and is composed of six
myocardial cells per hemisegment, except in A8, where only four are present
(Fig. 3A). Therefore, the total
number of the myocardial cells expressing Mef2 is 104
[(6x8+4x1)x2] (number of cells per hemisegment x
number of segments x 2 sides) (Fig.
3A). Two of the myocardial cells in each A2-A8 hemisegment express
svp but not tinman (SMC). Therefore, the total number of SMC
is 28 (2x7x2) (see Gajewski et
al., 2000; Lo et al.,
2002
). In the anterior two segments, all myocardial cells express
tinman (Fig. 9A),
which adds up to a total number of 76 TMCs
[(6x2+4x6+2x1)x2],
(Fig. 3C;
Fig. 9A) (Alvarez et al.,
2003). In embryos in which dap is expressed throughout the mesoderm,
thus blocking the last division, the number of myocardial cells is reduced in
T3-A1 (average 3.2 per hemisegment, n=20), as it is in the A2-A7
abdominal segments (two per hemisegment, n=76)
(Fig. 3B,D). In CycA
or Rca1 mutant embryos, however, which are normally blocked in cell
cycle 16, the number of TMCs in T3-A1 remains unchanged (six per hemisegment,
n=26), unlike in A2-A7 where the number of TMCs is reduced to half
(two per hemisegment, n=82) (Fig.
3E,F; Fig. 9B).
This suggests that in the anterior two segments, the TMC precursor divisions
are already complete after cycle 15, thus not affected in CycA and
Rca1 mutants, or the anterior myocardial lineages are different, as
suggested by a recent lineage study (Alvarez et al., 2003), or both. Whatever
turns out to be the case, these conclusions are consistent with recent
observation that the T3-A1 heart precursors are specified under homeotic
control that is distinct from that of the other abdominal segments
(Lovato et al., 2002
;
Lo et al., 2002
). Not only is
the myocardial cell number in T3-A1 unaffected in CycA or
Rca1 mutants, but as observed in a late stage embryos, these anterior
myocardial cells assemble into a tube as in wild type
(Fig. 3F). By contrast, the
fewer than normal abdominal myocardial cells do not align properly, suggesting
that a reduction in myocardial cell number adversely affects heart tube
morphogenesis.
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CycA or CycB mutants. By contrast, CycA;CycB
double mutants exhibit a reduction of TMC from six to three in 70% of observed
hemisegments (n=46; Fig.
4G,H). These data are consistent with the hypothesis that the
T3-A1 TMC progenitor divisions occur during cycle 15, which also fits with the
idea that the identity of these two cardiac segments is specified differently
(Lo et al., 2002; Alvarez et
al., 2003). Interestingly, the DA1 muscles are specified similarly in
CycA single or in CycA;CycB double mutants
(Fig. 1B; Fig. 4B,D), suggesting that Eve
progenitor specification is unaffected by arresting mitosis at cycle 15. This
is reminiscent of the observation made previously in stg mutants, in
which the cell division is arrested at cycle 14, the first post-blastoderm
division: up to two Eve progenitors are specified per hemisegment
(Carmena et al., 1998
;
Su et al., 1999
), one of which
will give rise to the DA1 muscle founder and the other to the immediate
precursor of two EPCs per hemisegment (Fig.
9C). Re-examining the cardiac phenotype in stg mutants we
found that the eve-expressing cells often appear in pairs, although
in some segments tinman- or eve-expressing progenitors fail
to form in this early arrest mutant. The pairs of eve-expressing
cells seem to adopt a muscle founder cell fate, as most of them express
Mef2 but not tinman in stage 13/14 embryos
(Fig. 4I,J). However, unlike
CycA;CycB double mutants, stg mutants show severe
segmentation and other patterning defects, presumably aggravated by the
paucity of cells that are formed, which precludes further interpretation of
the cardiac lineages.
Function of Numb and Notch in the asymmetric cardiac cell
lineages
Previous studies have shown that the asymmetric cell divisions in both the
Svp lineage and the Eve lineage are numb dependent and involve the
Notch pathway (Park et al.,
1998; Carmena et al.,
1998
; Carmena et al.,
2002
; Ward and Skeath,
2000
). In numb mutants, the number of myocardial cells in
segments A2-A7 is reduced from six to four per hemisegment
(Fig. 5A,B;
Fig. 9B), whereas the number of
myocardial cells in T3-A1 is not affected (data not shown) (Alvarez et al.,
2003). The reduction of myocardial cells is paralleled by an increase in EPC
number from two to four in 80% of 200 hemisegments counted, accompanied by a
loss of DA1 muscles (Fig. 5B;
Fig. 9B)
(Park et al., 1998
). By using
a Gal4 driver under the control of the mesodermal eve enhancer
(eme-Gal4) (Han et al., 2002
),
Numb and constitutively active N(icd), were expressed exclusively in the
mesodermal Eve lineage (Fig.
5C,D). In eme>numb embryos, no EPCs are formed, only
the DA1 muscles (Fig. 5C). By
contrast, no DA1 muscles are formed in eme>N(icd) embryos, and 60%
of 160 hemisegments exhibit an increase in EPC number from two to three or
four (Fig. 5D), supporting a
cell autonomous action of Numb and Notch in this lineage. Interestingly, the
remaining segments show no expression of eve, suggesting the
corresponding eve progenitors have not formed, as observed previously
with pan-mesodermal overexpression of N(icd)
(Park et al., 1998
). It is
likely that the variation in phenotype is the result of slight regional
differences in the onset of N(icd) expression: earlier expression eliminates
the progenitor, while later expression results in a sibling fate
transformation opposite to that observed in numb mutants.
|
As the function of Numb is thought to interfere with Notch activation in the sibling cell it segregates into (Fig. 9C), we wanted to examine if this activity is at the level of Notch itself or downstream in the pathway at the level of Su(H). We reasoned that if Numb acts at the level of activated Notch, overexpression of numb attenuates the N(icd) overexpression phenotype, but not that of Su(H)vp16. Indeed, it seems that increasing the level of Numb protein by eme-Gal4-mediated expression counteracts the effect of N(icd), in that more DA1 muscles and less EPCs form (Fig. 5G). By contrast, overexpression of numb together with Su(H)vp16 generates a phenotype similar to that of Su(H)vp16 overexpression alone (Fig. 5H), which suggests that Numb functions upstream of activated Su(H).
The phenotype of eme>Su(H)vp16 embryos is not as strong as with eme>N(icd), in that some DA1 muscles are still formed in some segments. This allowed us to address the question, whether or not DA1 muscle founders are siblings of EPCs, from a new angle: if they derived from the same precursor, DA1 muscle formation would never occur simultaneously with EPC duplication. By contrast, we did occasionally observe four EPCs and one DA1 muscle in the same hemisegment (Fig. 5F), consistent with an independent lineage of EPC and DA1 progenitors (Fig. 1F; Fig. 9C).
emeA-lacZ as a marker for sibling cell fates of the
mesodermal Eve lineage
A recent study suggests that the sibling cell of the EPC progenitor is the
DO2 muscle founder (Carmena et al.,
2002). However, muscle founders fuse with surrounding myoblasts
and eve expression disappears in the DO2 founder after the asymmetric
cell division is completed, which makes it difficult to follow cell fate
transformations between the proposed siblings. To circumvent this problem, we
took advantage of a mesodermal eve enhancer, emeA
(Han et al., 2002
), which
labels the EPCs and both the DA1 and DO2 founder nuclei, and founder
expression persists even after myotubes begin to form (but, unlike with
eve, the myoblast nuclei that have fused with the founders are
unlabeled; Fig. 6A). In
eme>dap embryos, both DA1 and DO2 founders form, but the number of
EPCs is reduced to half (Fig.
6B), similar to the phenotype observed with dap
overexpression throughout the mesoderm
(Fig. 1D). In the CycA
mutant, only the two muscle founders are present in each hemisegment
(Fig. 6C), suggesting that when
the asymmetric divisions of cycle 16 are blocked both progenitors adopt muscle
founder cell fate. In numb mutants, neither DA1 and nor DO2 founders
form; instead, four EPCs are present (Fig.
6D). We note that the occasional formation of a DO2 founder
(identified by position and absence of Eve protein) is always accompanied by
the presence of two instead of four EPCs (data not shown), consistent with a
lineage relationship between DO2 and EPC
(Fig. 1F;
Fig. 9C). Conversely, in
eme>numb embryos, two pairs of DA1 and DO2 muscle founders, but no
EPCs, are observed in 28% of 120 hemisegments counted
(Fig. 6E). In the remaining
segments, only one DO2 is observed accompanied always by two EPC with or
without a duplicated DA1 founder (data not shown). This suggests again that
DO2 rather than DA1 is related by lineage to the EPCs. Finally, N(icd)
expression in the mesodermal Eve lineage results in a loss of DO2 and DA1
founders accompanied by the concomitant formation of four EPC in 60% of 120
hemisegments counted (Fig. 6F),
or no eve or lacZ expression in the remaining segments (see
also Fig. 5D).
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DISCUSSION |
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Mesodermal Eve lineages
We show that the asymmetric cell divisions of the Eve lineage that
generates the EPC progenitors and DO2 founders are arrested in CycA
or Rca1 mutants, in which cell cycle 16 is blocked
(Knoblich and Lehner, 1993;
Dong et al., 1997
), but not in
the twi>dap embryos, in which the subsequent division of the EPC
progenitor is inhibited (cycle 17). Based on the effects of these genes in the
ectoderm, it is likely that CycA mutant blocks the progression of
cell cycle also in the mesoderm, but ectopic dap induces early exit
from the cell cycle. Therefore, in CycA mutants mitosis may be
blocked at a certain time point during development (such as at G2/M transition
of mitosis 16), but the ectopic dap may induce skipping of the last
division. It has been shown that the Numb crescent in the precursor P2, which
generates the DO2 founder and the EPC progenitor, appears at late stage 10 and
the division happens between late stage 10 and early stage 11
(Carmena et al., 1998
),
consistent with this being mitosis 16 of the mesodermal cells. Therefore, it
seems that many cells of the cardiac mesoderm go through three postblastoderm
cell divisions (mitosis 14-16), but some of them (such as the EPC lineage)
undergo an additional division (mitosis 17).
Two different models have been proposed for the mesodermal Eve lineages.
One model suggested that each EPC share a progenitor with a muscle founder
(Park et al., 1998). The other
model suggested that the two EPCs per hemisegment share a progenitor, which in
turn share a progenitor with one of the muscle founder cells (DO2), whereas
the other DA1 muscle founder derives from the second progenitor
(Carmena et al., 1998
). The
data presented in this paper strongly support the latter model (depicted in
Fig. 9). The most direct
evidence derives from pan-mesodermal dap overexpression, which
results in the formation of a single EPC, probably because of a block the last
division. By contrast, the first model predicts formation of either no EPC or
two EPCs, clearly not what is observed. These conclusions are also supported
by recent lineage tracing experiments (A. Alvarez and J. B. Skeath,
unpublished).
Myocardial lineages along the anterior-posterior axis
Recent studies have suggested that cardiac specification along the
anterior-posterior axis is under the control of homeotic genes
(Lovato et al., 2002;
Ponzielli et al., 2002
;
Lo et al., 2002
) (A. Alvarez
and J. B. Skeath, unpublished). For example, Svp myocardial cells are only
present in the abdominal segments of the heart, but not in the thoracic
segments (Gajewski et al.,
2000
; Lo et al.,
2002
); probably under the control of Antennapedia which
is active in these segments (A. Alvarez and J.B. Skeath, unpublished). In this
study we found that in addition to cell identity differences between the
anterior two and the posterior heart segments, the lineage of the T3-A1
myocardial cells are also distinct. In cycle 16 blocking CycA or
Rca1 mutants, the last cycle of myocardial divisions in T3-A1 is not
arrested unlike it is the case for the posterior myocardial cells. The
anterior myocardial progenitors may either undergo the last division during
cycle 15 or they may be less susceptible to a loss-of-CycA-function.
The first possibility is consistent with the observation that embryos with
overexpression of last division inhibitor dap, but not cycle 16
arrested CycA mutants, exhibit a reduction of TMCs in T3-A1.
Alternatively, it is possible that all T3-A1 myocardial lineages are
asymmetric, as the Svp lineages more posterior, except they all express
tinman, due to the lack of svp in these two segments
(Lo et al., 2002
). Thus,
blocking division in CycA mutants would not alter the number of TMC
in these two segments. Recent lineage tracing data are consistent with this
view (A. Alvarez and J. Skeath, unpublished), but further experiments are
needed to elucidate these lineages.
Extension of cardiac lineages
The second postblastoderm cell division of the mesodermal cells (mitosis
15) seems to be arrested if both CycA and CycB functions are
lost. In CycA;CycB double mutants, only two of the normally
six myocardial cells are formed in A2-A7 segments, one exhibiting TMC and the
other SMC characteristics. Therefore, we propose that in each hemisegment two
myocardial superprogenitors are specified
(Fig. 9): the TMC
superprogenitor (TSP) divides twice symmetrically, whereas the SMC
superprogenitor (SSP) first divides symmetrically and then asymmetrically.
Present and previous studies suggest there are probably five progenitors in
each hemisegment that give rise to 14 heart-associated cells (six myocardial
and eight pericardial): the TSP gives rise to four myocardial cells (two of
which are TLMC), the SSP generates two myocardial and two SOPC, the EPC
progenitor to two EPCs; the remaining four pericardial cells, two OPC and two
LPC, probably derive from two symmetrically dividing precursors, although
their lineage is not as well understood.
Asymmetric cell division and cell cycle progression
Asymmetric divisions have previously been studied in the context of cell
cycle progression in the Drosophila PNS and CNS (Vervoort et al.,
1997; Wai et al., 1999;
Tio et al., 2001
). In the PNS,
Notch activity is required for specification of a type I versus type II
neuronal fate. When sensory organ progenitor cell division is blocked in
stg- mutants, the undivided precursor adopts a type II
neuronal fate, whereas in numb;stg double mutants, a type I
fate is chosen (Vervoort et al., 1997). In the CNS, Notch is required for
specification of the sib cell fate versus the RP2 cell fate of the GMC1
asymmetric cell division. In Rca1 mutants, the undivided GMC1 adopts
a RP2 fate, whereas in numb;Rca1 double mutants, the
undivided GMC1 often adopts the sib cell fate
(Buescher et al., 1998
;
Wai et al., 1999
;
Lear et al., 1999
). Both
experimental outcomes are analogous to what we observe in CycA
mutants: the undivided P2 progenitor adopts a pericardial fate in the absence
of numb function instead of a myogenic fate in a wild-type
background. Taken together, these observations suggest that arrest of an
asymmetric cell division leads the undivided progenitor to adopt the fate of
the daughter cell that inherits Numb, and in the absence of Numb the
alternative fate is chosen.
Notch activity promotes autonomously a non-myogenic fate in
asymmetric lineages of the cardiac mesoderm irrespective of cell division
Previous studies suggested that Notch activity controls two distinct
processes during the specification of cardiac cell fates
(Park et al., 1998). First, it
is required to single initial progenitors out of a field of competence by
supporting the selection and inhibiting surrounding cells from adopting the
same fate (Culi and Modolell,
1998
). Subsequent to the progenitor specification, Notch is
required again for the specification of alternative cell fates of sibling
cells produced during asymmetric cell divisions (reviewed by
Jan and Jan, 1998
). In this
study, we have examined the cell autonomy of Notch, by using eme-Gal4 to drive
activated forms of Notch and Su(H) exclusively in the mesodermal Eve lineages.
We also used conditional ubiquitous expression of activated Notch to examine
its lineage-specific function in other cardiac lineages. We find that Notch is
required for specification of a non-myogenic fate in both the Eve and the Svp
lineages of the cardiac mesoderm. By contrast, activation or inhibition of the
Notch pathway did not affect cell fate decisions within the symmetric
lineages. This suggests a mechanism by which cell type diversity may be
increased during evolution by co-opting the Notch pathway during cell division
to distinguish between alternative fates of the daughter cells. The inability
of activated Su(H) to autonomously influence cell fates in symmetric cardiac
lineages further suggests that other factors or activities, not present in
symmetric lineages, are crucial for the asymmetric lineage-specific functions
of Notch and Su(H).
Interestingly, this influence of the Notch pathway on cell fate decision in
asymmetric cardiac Eve and Svp lineages is not altered when cell division is
arrested. Thus, cell division is not essential to distinguish between
alternative cell fates. The data also suggest that the default cell fate of a
asymmetrically dividing cardiac precursor in Drosophila is determined
to assume a myogenic fate, owing to Numb-mediated inhibition of Notch, unless
that fate is switched by the activation of target genes downstream of Su(H).
Moreover, in a double mutant of Notch and numb we would
expect to observe the same lineage phenotype as of Notch alone, i.e.
a myogenic cell fate, as the primary role of Numb is to inhibit Notch
signaling (see also Spana and Doe,
1996). Unfortunately, analysis of such double mutants is
complicated by the earlier role of Notch in lateral inhibition.
Another unresolved issue is the source of the Notch ligand that activates
signal transduction within asymmetric cardiac lineages. If the myogenic cell
were to produce the ligand for Notch activation in its pericardial sibling,
then the undivided progenitor would have to secrete its own Notch ligand. This
is unlikely, as production of the ligand is usually inhibited within the cell
that experiences Notch signaling (see Culi
and Modellel, 1998). In the asymmetric MP2 lineage of the
Drosophila CNS, for example, ligand production appears to be required
in cells outside the MP2 lineage (Spana
and Doe, 1996
). A similar scenario may be operating in the
asymmetric cardiac lineages.
Numb acts at the level of Notch in preventing Su(H) activation
Within the Eve lineages, Notch activation is mimicked by Su(H) fused to the
VP16, a potent transcriptional activation domain. Recent studies suggest that
in the absence of Notch activity, DNA-bound Su(H) prevents activators from
promoting transcription. When Notch ligands, such as Delta, bind to its
receptor, Notch is cleaved to produce an intracellular domain fragment,
N(icd), which is thought to enter the nucleus and interact directly with Su(H)
to recruit transcriptional co-activators and alleviate Hairless-mediated
repression, thus promoting transcription (for a review, see
Bray and Furriols, 2001;
Barolo and Posakony, 2002
). In
support of this model, we find that Su(H) overexpression can mimic Notch
activation only when linked directly to a transcriptional activator, but not
in its wild-type form when it presumably associates with co-repressors, such
as Hairless and Groucho (Barolo et al.,
2002
), that prevent Su(H)-dependent transcriptional activation in
the absence of Notch signaling.
The role of the PTB-containing, membrane-associated protein Numb in
preventing Notch activation in the nervous system is well established (for a
review, see Kopan and Turner,
1996; Jan and Jan,
1998
; Jafar-Nejad et al.,
2002
). To explore at which level Notch signaling is inhibited by
Numb in the cardiac lineages, we overexpressed numb simultaneously
with N(icd) or Su(H)vp16 within the mesodermal Eve lineages.
Excess Numb was able to counteract activated Notch but not activated Su(H)
function, suggesting that Numb can inhibit Notch activity after it has been
cleaved, possibly by preventing its nuclear translocation, but is unlikely to
prevent the transcriptional activator function of Su(H) directly. Recent data
suggest that Numb is involved in stimulating endocytosis of Notch, thus
removing it from the cell surface and inhibiting its function
(Berdnik et al., 2002
). It is
not clear, however, if this inhibition by endocytosis is at the level of the
entire Notch receptor, or (also) at the level of N(icd) after it is cleaved
off. Our experiments provide strong evidence that Numb can indeed interfere
with N(icd) function, but it remains to be determined if endocytosis is an
obligatory intermediate in this inhibition of activated Notch.
Notch activity may specify a pericardial cell fate in both
Drosophila and vertebrates by similar mechanisms
A recent study in Xenopus suggests that the Notch pathway may also
have a role in vertebrates in specifying pericardial and other non-myogenic
cell fates within the dorsolateral cardiogenic region of the anterolateral
plate mesoderm (Rones et al.,
2000). As in the Eve and Svp lineage of the Drosophila
heart, activation of the Notch pathway decreased myocardial gene expression
and increased expression of a pericardial marker, whereas inhibition of Notch
signaling resulted in an increase of cardiac myogenesis. Similar results were
obtained with an activated form of RBP-J [a vertebrate homolog of
Drosophila Su(H) fused to vp16, as in our study]
(Rones et al., 2000
). These
data indicate that the Notch pathway may play a role in the specification of
myocardial versus pericardial cell fates in both Drosophila and
vertebrates. This raises the question of whether the mechanism of Notch
mediated cell identity determination is also conserved between vertebrates and
flies. Because it is not yet known if (Numb-controlled) asymmetric cell
divisions are also involved in vertebrate heart development, the answer awaits
future studies. However, recent studies on the role of Numb during cortical
development suggest that it is likely to have a similar control function in
cell fate specification in vertebrates as it does in flies
(Shen et al., 2002
).
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ACKNOWLEDGMENTS |
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