Cutaneous Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Building 149, 13th Street, Charlestown, MA 02129, USA
Author for correspondence (e-mail:
laurel.raftery{at}cbrc2.mgh.harvard.edu)
Accepted 13 August 2003
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SUMMARY |
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Key words: BMP, Morphogen gradient, SMAD, Dorsal-ventral patterning, Drosophila
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Introduction |
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BMP signaling is complex in early embryos, requiring two BMP ligands, Screw
(SCW) and Decapentaplegic (DPP), and two type I receptor serine-threonine
kinases, Saxophone (SAX) and Thickveins (TKV) (reviewed by
Raftery and Sutherland, 1999).
Receptors and SMAD signal transducers are present in the oocyte, so that the
onset of signaling depends on zygotic transcription of ligand genes.
dpp is expressed over the dorsal 40% of the embryo during blastoderm
and gastrula stages (St. Johnston and
Gelbart, 1987
), whereas scw is expressed globally for a
short period during blastoderm cellularization
(Arora et al., 1994
).
Patterns of ligand RNA accumulation are broader than the BMP activity
gradient inferred from the pattern of dorsal fates (reviewed by
Podos and Ferguson, 1999). A
narrow band of dorsal midline cells become amnioserosa, the dorsal-most fate,
in response to high BMP activity (Ferguson
and Anderson, 1992a
; Wharton
et al., 1993
). Both ligands, DPP and SCW, are required for this
fate (Arora et al., 1994
;
Neul and Ferguson, 1998
;
Nguyen et al., 1998
). In
scw null or weak dpp mutants, the DV fate map shifts, so
that amnioserosa is lost, the dorsal ectoderm contracts, and ventral ectoderm
expands. In dpp null mutants, all ectoderm adopts the ventral
ectoderm fate.
The domain of BMP activity is narrowed through antagonism by Short
gastrulation (SOG), in collaboration with other proteins (reviewed by
Harland, 2001;
Ray and Wharton, 2001
). SOG is
a secreted BMP binding protein, which is distributed in the inverse pattern to
the proposed BMP activity gradient
(Srinivasan et al., 2002
).
Consistent with a role for SOG as a BMP antagonist, sog null embryos
have an expanded dorsal ectoderm (Ferguson
and Anderson, 1992b
; Francois
et al., 1994
). Surprisingly, sog null embryos lack the
expansion of amnioserosa predicted for a BMP antagonist. Instead, they
differentiate only a few amnioserosa cells. Genetic manipulations consistently
support a positive role for SOG in amnioserosa patterning
(Ashe and Levine, 1999
;
Decotto and Ferguson, 2001
).
This dual role led to a proposal that SOG directs ligand transport from
lateral to dorsal regions (Holley et al.,
1996
), a model that has received recent experimental support
(Eldar et al., 2002
;
Ross et al., 2001
).
The BMP activity gradient was interpreted from the patterns of BMP-directed
gene expression and terminally differentiated cell types. BMP target genes are
expressed in domains centered on the dorsal midline, with smaller expression
domains nested within the larger ones (Ashe
et al., 2000; Jazwinska et
al., 1999
). However, the pattern of target gene expression also
depends on Brinker, a transcriptional repressor that competes with
BMP-activated SMADs to regulate target genes
(Ashe et al., 2000
;
Jazwinska et al., 1999
). After
blastoderm, BMP activity negatively regulates brinker, limiting
expression to the ventral ectoderm domain. In addition, BMP activity
positively regulates genes that elevate BMP activity, including dpp
(Biehs et al., 1996
;
Zhang et al., 2001
). Thus,
BMP-dependent gene expression is a complex output of direct and indirect
responses. Assays to detect direct BMP responses are essential to understand
the mechanisms for spatial deployment of active BMPs.
SMAD proteins mediate the gene expression responses to TGFß
superfamily ligands (reviewed by Massague
and Wotton, 2000; Raftery and
Sutherland, 1999
). Two classes of SMADs collaborate to transduce
the intracellular signal from the transmembrane receptor complex to the
nucleus, the receptor-regulated SMADs (R-SMADs) and the common-mediator SMADs
(co-SMADs). For Drosophila BMP signaling, MAD is the key R-SMAD and
Medea is the only co-SMAD. MAD is directly phosphorylated in response to BMP
signaling. Phospho-MAD (P-MAD) accumulates in the nucleus, associates with
other transcription factors and binds DNA. Like the vertebrate co-SMAD, SMAD4,
Medea accumulates in nuclei of cultured cells only in the presence of a
phosphorylated R-SMAD. Co-SMADs also bind DNA and participate in transcription
regulatory complexes. Detection of signal-dependent SMAD responses, whether as
nuclear accumulation or phosphorylation, visualizes the pattern of active
responses to TGFß family signals. Here we find that patterns of co-SMAD
responses undergo two transitions during blastoderm and gastrula stages, to
form a step gradient of domains with different response levels. Formation of
the step gradient requires both BMP ligands and SOG. The evolving pattern of
BMP responses provides important insights into BMP-directed patterning of
dorsal fates.
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Materials and methods |
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To overexpress Medea we used either homozygous Ubi>Medea or
P{GAL4-prd.F} (prd>Gal4)
(Brand and Perrimon, 1993)
driving UAS>Medea.
For larval protein extracts, red mwh e Med1/Df(3R)KpnA, ca awd or Mad10 b pr/Mad12 b pr wandering third instar larvae were selected by their transparency.
For ventro-lateralized embryos, homozygous cactPD74 cn bw females were obtained at 18°C and mated to y1 w67c53 males at 25°C.
Germline clones were generated using the FLP-DFS system as described
(Chou and Perrimon, 1996). For
Medea, we generated clones in either
P{ry+t7.2=neoFRT}82B Med13
(Hudson et al., 1998
) or
P{ry+t7.2=neoFRT}82B Med8
(Wisotzkey et al., 1998
)
females, which were mated to Med13/TM3 or
Med8/TM3 males, respectively. Mad germline clones
were generated in P{ry+t7.2=hsFLP}12, y1 w*;
Mad10 P{ry+t7.2=neoFRT}40A females, which were
mated to Mad12/CyO males. There have been conflicting
reports on the fertility of females bearing germline clones homozygous for
Mad12 (Das et al.,
1998
; Wisotzkey et al.,
1998
). We tested the two previously used heat-shock protocols on
the two previously described FRT Mad12 strains; in each
case, no embryos were obtained with this allele. The cause for differing
results remains unknown.
Constructs
Medea constructs used the cDNA AF027729
(Wisotzkey et al., 1998). Two
were generated by PCR: pET28MedFL contains codons 56-782 (stop) cloned into
the SacI site of pET28a (Novagen). pGEX5MedC1 contains codons 399-782
(stop) in frame with the GST open reading frame (ORF) in pGEX5X-1
(Pharmacia).
UAS>Medea: An EcoRI-XbaI fragment containing
3' sequences from the cDNA in pBluescript was subcloned into the
EcoRI-XhoI sites of pUAST
(Brand and Perrimon, 1993),
using T4 DNA polymerase to blunt the XhoI and XbaI ends. The
5'EcoRI fragment of the cDNA was sub-cloned into the resultant
plasmid and sequenced. Transgenic flies were generated by the CBRC Transgenic
Fly Core.
Anti-Medea antiserum
Full-length Medea fusion protein was expressed from pET28MedFL as
recommended (Novagen). Bacterial extracts were subjected to SDS PAGE; fusion
protein was excised and electro-eluted. Rabbit antiserum was produced at
Poconos Rabbit Farm and Research Laboratory (Canadensis, PA).
Affinity purification followed established protocols
(Harlow and Lane, 1988), using
C-terminal fusion protein from pGEX5MedC1.
Immunohistochemistry
For most experiments, embryos were collected for 1 hour at 25°C, aged
for 2 hours 50 minutes at 25°C, prepared and fixed as described
(Wisotzkey et al., 1998).
Unpurified antiserum was pre-adsorbed at 1/1000 in PBSS (PBS pH 7.4 containing
0.1% saponin and 3% normal goat serum) against WT embryos collected for 1 hour
and aged for 1 hour 50 minutes at 25°C. Immunofluorescence experiments
gave the same results with either affinity-purified antiserum or pre-adsorbed
antiserum. We used pre-adsorbed anti-Medea antiserum for most experiments.
Primary and secondary antibodies were incubated with embryos in PBSS
overnight. Pre-adsorbed anti-Medea antiserum was used at 1/1000.
Anti-phospho-SMAD1 (PS1) (Persson et al.,
1998
) was used at 1/100. Prior to mounting in Vectashield (Vector
Labs), embryos were incubated with 0.2 µM ToPro3 (Molecular Probes) in
PBSS, and washed briefly. Images were collected on a Leica confocal microscope
at a fixed gain for each experiment. Gain-matched WT and mutant images were
paired and manipulated together in Adobe Photoshop.
Protein extracts and Western blotting
Embryos were dechorionated and homogenized in lysis buffer (PBS; 5%
glycerol; 0.1% Triton X-100) with protease inhibitors (Roche). Lysates were
centrifuged at 14,000 g for 10 minutes; supernatant was stored
at 80°C. Wandering third instar larvae were homogenized for 20
minutes on ice, then centrifuged for 30 minutes at 14,000 g.
Western blots were performed as described
(Li et al., 1999) with
affinity-purified anti-Medea and chemiluminescence (Tropix). Blots were
stripped (0.2 M glycine, 0.1% SDS, 1% Tween-20, pH 2.2) and re-probed with
either monoclonal anti-actin (Chemicon International) or monoclonal
anti-tubulin (Cedarlane Laboratories).
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Results |
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Nuclear co-SMAD is BMP-dependent in early embryos
Endogenous BMP signals stimulate only a fraction of cytoplasmic MAD to
accumulate in the nucleus (Dobens et al.,
2000; Newfeld et al.,
1997
). In contrast, nuclear accumulation of endogenous Medea was
detected in WT embryos (Fig.
1A, Fig. 2B,C). We
investigated whether this was a response to TGFß superfamily signals.
|
|
Patterns of SMAD responses are dynamic
In most blastoderm embryos, levels of nuclear Medea were indistinguishable
between dorsal and ventral cells (Fig.
2A). The earliest embryos in which dorsal cells had increased
nuclear accumulation of Medea were late in stage 5, between the end of
cellularization and the beginning of gastrulation
(Fig. 2B). At this stage,
nuclear accumulation was detected in a 32 cell-wide domain centered at the
dorsal midline. Within this domain, nuclear staining appeared consistent over
approximately 24 cells. At the edges, levels of nuclear Medea decreased over
approximately four cells. More laterally, the level of nuclear staining was
indistinguishable from that in ventral midline cells. Not all stage 5 embryos
had significant nuclear Medea, suggesting that this phase is brief.
In contrast, early gastrula embryos had a narrow stripe of approximately 5-7 cells with detectable nuclear Medea. Within this stripe, the central three cells had intense nuclear staining (Fig. 2C, Fig. 3A); nuclear staining dropped sharply across two cells at each edge. Thus, the domain of detectable co-SMAD response narrowed significantly, as levels increased at the dorsal midline.
In our experiments, the earliest PMAD staining was detected at the
beginning of gastrulation, co-incident with the narrow stripe of strong Medea
staining (Fig. 3B,D). More
sensitive assays detected an earlier broad domain of weak PMAD staining during
cellularization (Ross et al.,
2001; Rushlow et al.,
2001
), similar to the earliest nuclear Medea pattern. For PMAD,
the broad domain of weak response was detected during mid-cellularization, and
the transition to a narrow stripe of strong response was detected during late
cellularization (Rushlow et al.,
2001
), in both cases earlier than for Medea. Technical differences
between antibodies and staining techniques may contribute to this difference.
It may also reflect the time between receptor activation at the cell surface
and SMAD accumulation in the nucleus, which takes 15-20 minutes for activin
responses in Xenopus cells
(Bourillot et al., 2002
). Stage
5 cellularization spans 40 minutes
(Campos-Ortega and Hartenstein,
1997
).
SMAD-response patterns changed further during gastrulation (stages 6-7), a
period of approximately 20 minutes. For both nuclear Medea and PMAD, the
domain of most intense staining widened to include approximately 7-9 cells
(Fig. 3E,F,
Fig. 4). In the midline stripe,
nuclear Medea staining intensified, but maintained a sharp decline at each
edge. The stripe pattern persisted in the cephalic region
(Fig. 4A). Between the cephalic
furrow and the posterior furrow, the domain of intense staining became
irregular, as presumptive amnioserosa cells rearrange to accommodate the
extending germband (reviewed by
Campos-Ortega and Hartenstein,
1997). During mid- to late-gastrulation, dorsolateral domains with
low nuclear Medea were detected, which encompassed approximately 6-7 cells
beyond the intensely staining region (Fig.
4). These cells had a uniform distribution of Medea that was
distinct from the nuclear staining in more dorsal cells (compare
Fig. 4D,D' with Fig. 4B,B'). More ventral cells showed staining in the cytoplasm, with occasional speckles
staining the nuclei (Fig.
4C,C'). The speckles may reflect shuttling through the nucleus in
the absence of TGFß family signals
(Pierreux et al., 2000
).
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In summary, Medea responses to BMP activity form three distinct patterns, beginning with a weak dorsal response at cellular blastoderm. At the onset of gastrulation, the pattern narrows to a strong dorsal midline response. The midline response intensifies and spreads during gastrulation, when flanking dorsolateral domains develop weak Medea responses. This step gradient of responses does not include as many lateral cells as the initial blastoderm response.
Sharp transitions in BMP activity correlate with pattern
boundaries
To test the correlation between the domain of intense SMAD responses and
the position of dorsal pattern markers, we examined the cell division 14
mitotic domains 1, 3 and 5 of the cephalic region
(Arora and Nüsslein-Volhard,
1992). Mitotic domains are spatially restricted regions of cells
that undergo synchronous mitosis after cellular blastoderm
(Foe et al., 1993
). Condensed
mitotic chromosomes were detected with ToPro3 DNA dye in embryos stained for
Medea or PMAD. Mitotic domain 3 precisely straddled the stripe of peak SMAD
response; the dorsolateral domains 1 and 5 abutted its edges
(Fig. 5A,B). Mitotic domains 3
and 5 remained abutted to the narrower midline response in 1Xdpp
embryos (Fig. 5C) and also to
the wider midline response in 4Xdpp embryos
(Fig. 5D-PMAD, and data not
shown-Medea). A broader domain 3 straddled the broader SMAD response stripe of
4Xdpp embryos. Thus, the edges of the midline stripe define
transitions between BMP activity levels that direct different cell fates.
|
To investigate the role of DPP, we stained embryos produced by dppH46/CyO23, P{dppH+} adults. Within the resultant population of stage 6 embryos, some lacked nuclear Medea (Fig. 6A). Similarly, PMAD staining was absent in some embryos; occasional staining in primordial germ cells of such embryos may be artifactual (Fig. 6B). Late-gastrula dpp mutants were visibly distinct, and lacked SMAD responses (Fig. 5E). Thus, DPP is necessary for both SMAD responses.
|
The level of peak response is sensitive to dpp dosage
DV patterning is highly sensitive to DPP levels. Loss of one copy of
dpp is lethal, causing a range of defects in head skeleton and
amnioserosa patterning (Irish and Gelbart,
1987; Wharton et al.,
1993
). Increased dpp dosage leads to more amnioserosa
cells (Wharton et al., 1993
).
Thus, we investigated the effects of dpp dosage on the patterns of
SMAD responses. Contrary to a previous report
(Dorfman and Shilo, 2001
), we
found a variable dorsal midline response in most dpp-/+ embryos.
For this experiment, we examined both PMAD and Medea staining in embryos from a mating of dppH46/P{dppH+} with WT. Resultant embryos were either dppH46/+ (1Xdpp) or P{dppH+} plus the two endogenous copies of the gene (3Xdpp). Wild-type embryos were stained and imaged in parallel (2Xdpp).
At stage 6, when the WT response domain narrows and nuclear Medea
intensifies at the dorsal midline, all embryos from this mating had a narrow
stripe of activated SMAD that covered a similar domain to WT (data not shown).
Thus, neither 1Xdpp nor 3Xdpp altered the transition to a
narrow midline response. However, the strength of the response was much lower
in 1Xdpp embryos by stage 7 (Fig.
7B). At the end of gastrulation, 3Xdpp and 1Xdpp
embryos were easily distinguished (Fig.
7A-F). 3Xdpp embryos had a broader midline response
domain than WT, particularly in the cephalic region. 1Xdpp embryos
had narrower and weaker midline responses, as measured by either nuclear Medea
(Fig. 5C, Fig. 7F) or PMAD
(Fig. 7D). At stage 8, some
1Xdpp embryos had a weak midline response
(Fig. 7F); in others, it was
undetectable (Fig. 7D). The
variable midline response in 1Xdpp embryos fits the terminal
phenotypes. All dppH46/+ embryos have defects in the head
skeleton and head involution; reduced amnioserosa is less penetrant
(Wharton et al., 1993).
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Increased Medea expands the amnioserosa
Levels of nuclear Medea in dorsal regions correlate well with the DV
patterning outcome, supporting the model that nuclear SMAD activity is the
functional output of BMP signaling. Studies of cultured cells indicate that
increased SMAD protein levels can increase target gene expression (reviewed by
Derynck et al., 1998). If the
level of nuclear SMAD determines cell fate, then increased Medea levels should
expand the most dorsal fate, the amnioserosa. We tested this with
overexpressed Medea.
To assess the number of cells that acquire the amnioserosa fate, we counted Krüppel-positive amnioserosa nuclei in stage 13 embryos. Overexpression throughout development, in ubi>Medea embryos, gave 224±6 (average±s.e.m.; n=11) Krüppel-positive nuclei, significantly more than the WT 159±5 (n=10; P<0.01 by the two-tailed t test). ubi>Medea embryos also had subtly increased dorsal midline P-MAD staining (Fig. 5F). Thus, increased levels of co-SMAD can expand the domain of a BMP-induced cell fate. In an initial assessment of the critical period for this effect, we overexpressed Medea after gastrulation, in prd>Gal4; UAS>Medea embryos. Increased expression was detected at stage 9 (data not shown), but the number of amnioserosa cells was not significantly different from WT.
SOG shapes the SMAD response gradient
SOG has a complex role in DV patterning, for it both antagonizes dorsal
ectoderm patterning and promotes the amnioserosa fate
(Decotto and Ferguson, 2001).
Patterns of SMAD responses should indicate whether the positive effect of SOG
is transmitted through the BMP signaling pathway, but reports of PMAD patterns
in sog mutants are conflicting
(Dorfman and Shilo, 2001
;
Ross et al., 2001
;
Rushlow et al., 2001
). To
examine co-SMAD responses, we selected a strong allele,
sogY506, and a molecular null, sogU2
(Francois et al., 1994
). The
two alleles gave similar results, assessed in stage 6 embryos.
For both Medea and PMAD staining, we detected three classes of embryos produced by heterozygous adults. One class appeared WT, and included the +/+ embryos (sogU2: 19/43; sogY506: 3/16) (Fig. 8A,B). A second class showed an intense dorsal stripe of staining that was variably broader than WT (sogU2: 19/43; sogY506: 10/16) (Fig. 8C,D), and probably included sog+/ females. The third class lacked the dorsal stripe of intense staining (sogU2: 5/43; sogY506: 3/16) (Fig. 8E,F). These embryos had a broad dorsal domain with low levels of nuclear Medea (Fig. 8E) or weak PMAD staining (Fig. 8F), and were most probably sog/.
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Discussion |
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BMP activity directs nuclear accumulation of co-SMAD in early
embryos
These in vivo studies validate the molecular model for signal-dependent
nuclear accumulation of co-SMAD. Nuclear accumulation of Medea requires both
competence to oligomerize and an R-SMAD, MAD. Nuclear accumulation is signal
dependent, requiring both BMP ligands, DPP and SCW. Conversely, all cells
accumulated nuclear Medea in the presence of constitutively active TKV
receptor. At these stages, any independent contribution from activin-like
signals is below the detection limit.
Furthermore, levels of Medea determine the strength of BMP responses at
these stages. Medea overexpression led to expansion of the dorsal-most fate,
with increased numbers of amnioserosa cells. Signal-dependence for nuclear
accumulation was retained (data not shown). Decreased Medea exacerbates loss
of amnioserosa from reduced DPP levels
(Raftery et al., 1995).
The intensity of Medea staining was surprisingly sensitive to signal activity. However, our tests showed that steady-state levels of Medea were unaffected by the level of BMP activity. Our antibodies appeared highly sensitive to a Medea conformation that is prevalent in the nucleus, most probably an active SMAD complex. This sensitivity makes nuclear Medea an excellent assay to distinguish spatial patterns of endogenous BMP activity.
SMAD responses reveal dynamic patterns of BMP activity
In WT embryos, two transitions in the distribution of BMP activity were
evident (Fig. 9). Many cellular
blastoderm embryos lacked detectable levels of nuclear Medea, but a few had
low levels of nuclear Medea in a broad dorsal domain, with little gradation
(Fig. 9A). From the proportion
of cellular blastoderm embryos with this pattern, it appears to be brief.
These data parallel reports of broad, weak PMAD staining during
mid-cellularization (Ross et al.,
2001; Rushlow et al.,
2001
), except that nuclear Medea is detected later and in a
broader pattern. The time lag between the earliest reported detection of PMAD
and our detection of nuclear Medea probably stems from a combination of
technical differences and the time necessary for nuclear accumulation. In sum,
initial BMP activity is weak and distributed broadly in dorsal regions. Low
BMP activity at this phase is required to maintain the early phase of
zen expression (Rushlow et al.,
2001
).
|
A third response pattern arose during mid-gastrulation; dorsolateral
domains of cells exhibited low levels of nuclear Medea
(Fig. 9C). Response levels
remained high in the dorsal-most cells, even as they moved laterally during
gastrulation (Fig. 3E,
Fig. 4A). Levels fell off
rapidly over a few cells on either side, with a sharp transition to flanking
plateaus of weak responses. The subcellular distribution of Medea was
unchanging in ventral and ventrolateral cells. The full BMP response domain
did not extend as far ventrally as it did during blastoderm, even though many
dorsal cells move laterally during germband extension
(Campos-Ortega and Hartenstein,
1997). Thus, the lateral-most cells with responses at blastoderm
had decreased responses during gastrulation.
In sum, the dorsal midline stripe of SMAD responses corresponds to a steep
BMP activity gradient, with thresholds that correlate with patterning markers.
The edges of the Medea peak response correlated precisely with the position of
dorsal cephalic markers during stage 8, the cycle 14 mitotic domains 1, 3 and
5. The second phase of zen expression occurs in cells with peak PMAD
responses at the end of stage 5 (Rushlow
et al., 2001). Flanking cells with lower PMAD levels correlate
with the broader expression domain for the BMP target genes tailup
and u-shaped (Ashe et al.,
2000
; Rushlow et al.,
2001
). The full Medea response domain correlates approximately
with the expression domain for u-shaped and extends into the
presumptive dorsomedial ectoderm. The sharp transitions in SMAD response
levels predict expression boundaries for BMP-responsive genes.
Similarly, in the wing primordium, a BMP gradient creates sharp transitions
in PMAD levels, which match gene expression boundaries
(Tanimoto et al., 2000;
Teleman and Cohen, 2000
).
However, BMP activity is modulated by different mechanisms in this tissue.
dpp is expressed in a narrow stripe at the center, and ligand spreads
to nearby cells over a period of hours. In contrast, the early embryonic BMP
activity gradient forms rapidly, and is narrower than the expression domains
for dpp and scw. Extracellular binding proteins form the
embryonic BMP activity gradient.
The dorsal midline gradient is shaped by DPP and SOG
The final width of the midline peak response is sensitive to gene dosage
for both dpp and sog. It is broader when dpp dosage
is increased (Fig. 5D,
Fig. 7A), and narrower with
only one copy of dpp (Fig.
5C, Fig. 7F,
Fig. 9E). Similarly, the width
of the stripe was broader, but more variable, when sog levels were
reduced (Fig. 8C,D,
Fig. 9D). The response domain
was broadest in sog null embryos
(Fig. 8E,F); however, the level
of response was significantly reduced. This was distinct from the effect of
increased dpp dosage, in which the response domain was broader, but
normal SMAD response levels were achieved or exceeded.
The role of SOG as both a short-range inhibitor and a long-range
potentiator of dorsal patterning led to a proposal that SOG transports BMP
ligands from lateral regions to the dorsal midline
(Holley et al., 1996).
Biochemical analyses suggest mechanisms for SOG-BMP binding and release
(reviewed by Harland, 2001
;
Ray and Wharton, 2001
).
Computational analysis defined conditions under which transport could occur
with these mechanisms (Eldar et al.,
2002
). The transition from weak, broad SMAD responses to narrow,
strong responses is consistent with concentration of BMP activity at the
dorsal midline, and the loss of this transition with loss of SOG is consistent
with a SOG-dependent transport model. However, there are significant
differences between our results and the assumptions used to develop the
computational model. These include the presence of a midline SMAD response in
dpp/+ embryos and the sensitivity to reduced sog
dosage. It will be important to refine future computational models to fit the
complete set of BMP response data.
Altered activity at different phases affects different tissue
boundaries
Both BMP ligands, DPP and SCW, were required to form the dorsal-midline
gradient. However, scw mutant embryos retain a small amount of dorsal
ectoderm, with concomitant expansion of ventral ectoderm
(Arora et al., 1994).
Surprisingly, the weak dorsolateral Medea response is lost in scw
embryos. We conclude that the full Medea response domain encompasses the cell
fates that are lost in scw mutants, amnioserosa and dorsomedial
ectoderm (Arora and Nüsslein-Volhard,
1992
). It appears that dorsal cells can acquire a dorsolateral
fate without gastrula BMP activity.
Mutants with expanded ventral ectoderm show reduced SMAD responses during
the first phase of BMP activity. PMAD was not detected in blastoderm
tld embryos (Ross et al.,
2001). Homozygotes for moderate dpp alleles have lower
PMAD levels during blastoderm (Rushlow et
al., 2001
). Conversely, sog embryos have a slightly
expanded PMAD response during blastoderm
(Rushlow et al., 2001
), and a
slight expansion of dorsal ectoderm
(Ferguson and Anderson,
1992b
). Thus, BMP activity during blastoderm positions the
boundary between dorsal and ventral ectoderm.
Mutations that shift the boundary between amnioserosa and dorsal ectoderm
show altered SMAD responses in the third phase of BMP activity, the
dorsal-midline gradient. dpp/+ embryos had variable reductions
in midline SMAD responses (Fig.
7) and in the number of amnioserosa cells
(Wharton et al., 1993).
Strikingly, sog null embryos have little amnioserosa and a strong
reduction in SMAD response levels during gastrulation
(Fig. 8). Thus, SMAD response
levels during gastrulation are critical for amnioserosa specification.
Multiple rounds of BMP signaling pattern DV fates
Taken together, these data suggest a multi-step model for DV patterning of
the embryonic ectoderm, incorporating aspects of the two previous models. In
the previous gradient model, ectodermal fates are subdivided simultaneously by
a continuous BMP gradient involving DPP and SCW
(Ferguson and Anderson,
1992a). In the successive cell-fate decision model, amnioserosa is
specified by dorsal-midline DPP+SCW activity, and the dorsal ectoderm by DPP
alone at stage 9 (Dorfman and Shilo,
2001
).
Instead, we propose that the blastoderm phase of weak BMP activity
establishes a dorsal ectoderm domain. As discussed above, mutations that shift
the boundary between dorsal and ventral ectoderm also have altered SMAD
responses at this stage. It is at this stage that SMADs compete with Brinker
to regulate the first phase of zen expression
(Rushlow et al., 2001).
Furthermore, this early signal maintains BMP activity, for the late-blastoderm
domain of dpp expression is set by competition between BMPs, SOG and
Brinker (Biehs et al., 1996
;
Jazwinska et al., 1999
). BMP
activity subsequently maintains the dorsal boundary for brinker
expression (Jazwinska et al.,
1999
). Thus, BMP activity at blastoderm defines a dorsal domain
where dpp is expressed and brinker is not.
After cellularization is complete, a step gradient of BMP activity subdivides the dorsal region into amnioserosa, dorsomedial ectoderm and dorsolateral ectoderm. Peak activity levels determine the amount of amnioserosa. Flanking shoulders of weak activity specify the dorsomedial ectoderm. We propose that the dorsolateral ectoderm experiences a transient BMP response during late blastoderm, but little or no response during gastrulation. In sum, the dorsal-midline gradient of BMP activity specifies at least three cell fates.
BMP activity in the dorsal ectoderm does not end with germband extension.
During stage 9, PMAD is detected throughout the dorsal ectoderm and
amnioserosa, and might finalize determination of dorsal ectoderm fates
(Dorfman and Shilo, 2001). DPP
expression within the dorsal ectoderm contributes to combinatoral regulation
of gene expression patterns in subsets of dorsal ectodermal cells
(Reim et al., 2003
). However,
the ventral boundary of dpp expression in the stage 9 dorsal ectoderm
must be defined by earlier events.
Implications for molecular mechanisms
The step gradient of SMAD responses is maintained during the morphogenetic
movements of gastrulation and germband extension. The peak response is
maintained only in cells that initially resided at the dorsal midline, even
though ventral ectoderm moves to a dorsal position during stages 7 and 8. The
BMP activity gradient is thought to form by diffusion in the perivitelline
fluid; however, dorsal cells `remember' their BMP exposure as they move
laterally [perhaps similar to memory of signal strength as discussed in
Bourillot et al. (Bourillot et al.,
2002)]. It is probable that the ligand distribution is established
prior to the time that peak SMAD responses are detected, and activity persists
through cell biological mechanisms. For example, ligand may bind to the
extracellular matrix (Fujise et al.,
2003
), so that it remains associated with dorsal cells.
Alternatively, receptor-ligand complexes may continue to signal following
endocytosis, as described for TGFß
(Penheiter et al., 2002
).
Understanding the intracellular modulation of BMP responses will be important
to understand how extracellular morphogen gradients are translated into a
stable pattern of cell fates.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Program in Genetics and Genomics, Hospital for Sick
Children, Toronto, Ontario M5G 1X8, Canada
Present address: Veterinary Research Institute, Hungarian Academy of
Sciences, Budapest H-1143, Hungary
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