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First published online 9 July 2008
doi: 10.1242/dev.017814
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1 Department of Pathology, Stanford University School of Medicine, 300 Pasteur
Drive, Stanford, CA 94305, USA.
2 Department of Genetics, Howard Hughes Medical Institute, Harvard Medical
School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
* Author for correspondence (e-mail: jaxelrod{at}stanford.edu)
Accepted 18 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, Hedgehog, Wingless, Embryo, Patterning
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
consist of groups of cells that coordinate patterning by inducing the fate of
their neighbors. According to the `organizer model', the inductive signal
carries information that dictates both the position and the nature of the
cell. Studies in various systems have led to the identification of a number of
signaling pathways, mediated by secreted ligands that bind to specific
receptors and appear to play instructive roles in determining cellular
identity and patterning, in conjunction with cellular history that provides
competence to interpret signals in specific ways. Some ligands are thought to
execute these functions in a concentration-dependent fashion, determining
different cell identities at different levels of ligand concentration. A key
feature of this model is that signals induce cell identities. Throughout this
discussion, by `induction' we mean the eliciting of a cell state different
from that in which a cell existed prior to reception of the signal.
Many secreted signals and the components of their signaling pathways have
been identified in genetic screens conducted in the Drosophila embryo
(Nusslein-Volhard and Wieschaus,
1980; Perrimon et al.,
1989; Perrimon et al.,
1996). This amenable genetic system provides an effective paradigm
to investigate patterning. In particular, various types of denticles and hairs
on the larval cuticle provide markers to assess patterning. Distinct models
have emerged to explain ventral and dorsal epidermal patterning. Wg and Hh are
thought to control the patterning of the ventral epidermis by acting on
secondary secreted signals, such as the EGF ligand Spitz, that constitute a
system of induction by relay (Alexandre et
al., 1999). In the dorsal epidermis, Hh has been proposed to
induce directly and pattern several cell types posterior to the Hh-expressing
cells, one of them being distant from the source of secretion
(Heemskerk and DiNardo, 1994).
Because the data were interpreted to indicate that Hh induces two different
cell types at different threshold concentrations, Hh was described as a
morphogen. From this first proposal of Hh acting as a morphogen, the
Drosophila embryo has provided an elegant system with which to
investigate how inductive mechanisms control patterning.
Positioning of the ligand secreting cells is the primary determinant of
cell identity in these models. A single row of cells per segment expresses Wg
(Baker, 1987), followed
posteriorly by two rows that express Engrailed (En)
(Fjose et al., 1985;
Kornberg et al., 1985) and
secrete Hh (Tabata and Kornberg,
1994; Taylor et al.,
1993). The Wg and En cells form what has been referred to as the
parasegmental organizer, by analogy to the Spemann organizer and because they
form a boundary that does not coincide with the segmental boundary
(Martinez-Arias and Lawrence,
1985). The purpose of the segmentation cascade is to specify the
location of the parasegmental organizer that in turn was proposed to induce
cell fates that give rise to terminally differentiated cells.
The segmentation cascade involves the sequential action of several gene
families that define transient domains along the anteroposterior axis
(Nusslein-Volhard and Wieschaus,
1980). The pair rule genes control the positioning of the Wg- and
Hh-secreting cells: sloppy paired (slp) activates
wg transcription (Cadigan et al.,
1994) and even skipped and fushi tarazu activate
the transcription of en (DiNardo
and O'Farell, 1987; Lawrence
et al., 1987; Macdonald et
al., 1986). Positive feedback between the two signal secreting
cells ensures their mutual stabilization at the correct location
(van den Heuvel et al., 1993).
The position of the parasegmental boundary is therefore defined by the pair
rule genes, and maintained by Wg and Hh. According to the current model, the
spatial information carried by the pair rule genes is superseded by that
provided by En, Wg and Hh, which control intrasegmental patterning. En, Wg and
Hh are therefore called segment polarity genes. In this model, signaling by Wg
and Hh simultaneously provides spatial information and cell fate
specification.
To develop a more precise understanding of this system, we analyzed the
development of the groove cells. They differentiate immediately posterior to
the En- and Hh-expressing cells, and their differentiation is Hh dependent
(Larsen et al., 2003). We
identified Odd skipped (Odd) as a marker of groove cell identity, which
allowed us to monitor temporally the influence of Wg and Hh on the development
of the groove cells and their neighbors. We report data most consistent with
the idea that the groove identity is specified several hours before grooves
differentiate. Wg patterns the En stripe of cells by maintaining En,
antagonizing a transition towards a more posterior identity. The range of Wg
signaling determines the width of the En-expressing domain, and therefore the
position of the segmental boundary. The cells beyond the reach of Wg, and
therefore beyond the boundary, adopt the groove identity. In a distinct
subsequent step, Hh, which is secreted from the En cells, is required to
refine the groove cell pattern by blocking another transition towards a more
posterior identity. We propose that, in this tissue, contrary to the cell fate
induction model, signaling acts by blocking transitions of cell identity
rather than by instructing cell identity. Cell diversity is generated by a
progression of cell identity from one to the next, towards a default fate. The
patterning signals act by maintaining spatially selected cells in specific
intermediate identities until differentiation occurs.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Fly strains
OreR was taken as wild type. opa8,
run3, odd5, odd01863, hhAC,
ptcIN, nkd2 and UAS-yanact are
from the Bloomington Drosophila Stock Center. Trh-lacZ
(1eve1) was obtained from M. Krasnow, Cadherin-GFP from H. Oda,
En-Gal4 and UAS-hh from the Perrimon laboratory.
Antibodies
Anti-Odd was a generous gift from Jim Skeath, anti-Ptc from Pascal Therond
and anti-Slp from John Reinitz. Anti-aPKC was from Santa Cruz Biochemical
(sc-216) and mouse anti-β-galactosidase was from Promega. Monoclonal
antibodies against En, Ena and Dlg, developed by C. Goodman, and Wg, Cadherin,
Crb and Yan developed, respectively, by S. Cohen, T. Uemura, E. Knust and G.
Rubin were obtained from the Developmental Studies Hybridoma Bank.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(Fig. 1C,D) and atypical
Protein Kinase C (aPKC) (Wodarz et al.,
2000) (Fig.
1E''') are enriched at the apical circumference of the groove
cells at embryonic stage 12, when the epithelium starts folding (see Fig. S1
in the supplementary material). When the groove is fully formed, at stage 13,
the actin-binding molecule Enabled (Ena) is enriched at the junctions
separating groove cells, whereas it is detected only at the vertices in
non-groove cells (Grevengoed et al.,
2001) (Fig.
1E'). In the fully deepened grooves at stage 14, the dorsal
epidermis contains a single row of about 19 groove cells that terminates with
a groove slit, a several cell wide cluster of 8-10 cells in the ventral part
of the lateral epidermis. No groove cells are present in the ventral epidermis
(Fig. 1D)
(Martinez-Arias, 1993). Here,
we focus on the patterning of the single dorsal row of groove cells.
An additional marker, Odd
(Nusslein-Volhard and Wieschaus,
1980), is specifically expressed in all groove cells
(Fig. 1B-D,F). Odd is a
zinc-finger transcription factor (Coulter
et al., 1990) known for its pair rule function
(Coulter and Wieschaus, 1988),
but has also been reported to have gap and segment polarity properties
(Saulier-Le Drean et al.,
1998). Odd is expressed in seven stripes at blastoderm stage, and
in 14 stripes when the germ band elongates
(Coulter et al., 1990).
Significantly, we can follow the Odd protein expression pattern in fixed
embryos from early stages (Fig.
2A) until groove morphogenesis. To determine whether Odd
expression correlates with groove cell identity, we analyzed groove formation
in pair rule mutants, in which anteroposterior patterning defects result in
overall spatial disorganization of cell types. In both odd paired
(Fig. 2B,B',B''')
and runt (Fig.
2C,C') mutant embryos, we find that Odd-expressing cells
also express Ena (Fig.
2B'') and Crb (Fig.
2C') in the characteristic groove cell patterns, and form an
indentation in the epidermis (Fig.
2B'), although a full groove is not always present. In
odd hypomorphic mutant embryos, the remaining Odd protein is strictly
correlated with remaining groove cells
(Fig. 2D,D'). Similarly,
in other conditions that modify the number or position of Odd cells, there is
a strict one-to-one correspondence between Odd expression and groove (see
Fig. 4C'',F'').
Notably, in odd hypomorphic mutants, segments lacking Odd expression
do not make grooves. We conclude that Odd expression faithfully marks groove
cell identity after stage 12. As the expression of Odd precedes groove
morphogenesis by several hours, we wondered whether the 14 Odd stripes present
at germ band extension represent an earlier specification of the groove cell
identity.
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) to track cells by live imaging, we found that the epidermal
surface lost as the tracheal placode invaginates is replaced by convergence of
the cells dorsal and ventral to the placode
(Fig. 3B,C; see Movie 1 in the
supplementary material). Although we do not have a live marker for Odd
expression, two cell types could be identified by their morphological
features: the tracheal cells that invaginate at stage 10 and the groove cells
that adopt their rectangular shapes and align at the bottom of the grooves.
Tracing backwards, the progenitors of both the tracheal cells and the groove
cells could be identified, revealing that the single row of dorsal groove
cells originates exclusively from cells dorsal to the placode, in the region
where Odd is expressed, but not from cells anterior to the placode, where En
is present. We conclude that the groove cells originate from the Odd cells
that were previously dorsal to the tracheal placode. These Odd-expressing
cells are likely to be pulled by the invaginating tracheal cells to occupy the
space left by their invagination. Strikingly, the En cells respect the
segmental boundary and are not pulled posteriorly by the invaginating trachea,
indicating that the segmental boundary is effective before segmental groove
morphogenesis and before the border between the En-and Odd-expressing cells
straightens.
This result contradicts a previous report that the groove cells originate
from the posterior-most En cells of each segment
(Larsen et al., 2003). In the
previous study, progenitors of En cells were marked using En-Gal4 to drive
expression of cytoplasmic horseradish peroxidase (HRP), and HRP staining was
observed adjacent to groove cell nuclei, suggesting that the groove cells may
have derived from cells that had recently expressed En. We repeated this
experiment, using En-Gal4 to drive UAS-APC2::GFP to mark the cytoskeleton of
En cells. We found that at stage 14, many En cells extend processes labeled
with APC2::GFP that can reasonably be mistaken to be within the Odd cells (see
Fig. S2 in the supplementary material). Second, a minority of cells posterior
to the En stripe express GFP (see Fig. S3 in the supplementary material), but
that expression is not restricted to the groove cells (see Fig. S4 in the
supplementary material), and time lapse imaging shows that it does not
decrease with time, implying a non-physiological expression due to the Gal4
system rather than residual expression of GFP from a cell that has lost En
expression. We conclude that the groove cells originate from the
Odd-expressing cells and not the En cells.
Odd expression is refined during and after tracheal invagination
Interestingly, a cell division occurs during the morphogenetic movement
accompanying tracheal invagination, and we observed that some sisters of
Odd-expressing groove cells did not become groove cells
(Fig. 3B,C; see Movie 1 in the
supplementary material), indicating that the groove cells are selected from
among a larger Odd-expressing precursor population. Examination of Odd
staining in fixed embryos confirms that the number of Odd-expressing cells
decreases: two rows of Odd cells are present during germ band retraction, and
only one remains at stage 14. By following lacZ expression in
Odd-lacZ embryos, we verified that only the cells that end up
adjacent to the En stripe retain Odd expression, while their posterior
neighbors lose Odd expression during stage 13, keeping only the perduring
β-galactosidase (Fig.
3F,F',F''). Pair rule mutants provide additional
evidence that maintenance of Odd expression depends on the En cells. In these
mutants, the territories of En and Odd are no longer in regular stripes
(Fig. 3D,D',E); yet, as
in wild type, Odd is expressed in regions adjacent to En cells, still in broad
territories several cells wide at stage 12, but soon afterwards, exclusively
in single cell wide stripes that are immediately adjacent to the En cells.
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Early Wg patterns En and Odd expression, and defines the segmental boundary
The presence of Odd in the ancestors of the groove cells suggests that the
groove identity may be specified long before the differentiation of the
groove. We therefore examined the earlier specification of Odd-expressing
cells and its effect on groove differentiation. Regulation of the seven
stripes of Odd expression before germ band elongation has been studied, but
nothing is known about the mechanisms controlling the 14 stripes present
during germ band elongation. Two Odd homologues, Bowl and Drumstick, are also
expressed in 14 stripes located posterior to the En stripes, and have been
shown to be repressed by Wg signaling
(Hatini et al., 2005). We
therefore asked whether Wg controls the formation of the 14 Odd stripes. We
find that Odd expression expands at the expense of En expression when Wg
function is impaired, indicating that Wg inhibits Odd expression as the 14
stripes of Odd are established (Fig.
4A,B). At the end of germ band elongation, the 14 Odd stripes
appear broader in wgIL114 mutants, and, as expected, only
a few En cells remain. naked mutant embryos, in which Wg signaling is
derepressed (Zeng et al.,
2000), display the opposite phenotype
(Fig. 4D,E): the En stripes are
wider, and only a few Odd cells are present. Thus, Wg signaling controls the
allocation of cells into the En and Odd identities. Wg therefore patterns the
position of the segmental boundary that lies between these two cell types, and
the boundary is respected during trachea invagination, as Odd cells are
displaced along the boundary, but cells on either side do not pass from one
side to the other (Fig.
3B,C).
The regulation of Odd by Wg influences groove development. The later expression of Odd in wg mutants is highly dynamic. As En expression disappears, the broad domains of Odd, in 14 stripes at stage 9, give way to a pattern in which all the dorsal and ventrolateral cells of the trunk express Odd, forming two longitudinal stripes spanning the length of the trunk at stage 11 (Fig. 4B). However, by stage 12, Odd has disappeared, except in close proximity to the few small groups of En cells that remain. These exceptional small En/Odd territories express groove markers in the Odd cells and form localized indentations centered on the Odd cells (Fig. 4C-C''). nkd mutants also display a few remaining Odd cells, and these too differentiate as groove cells. In these mutants, no grooves are present at the border of the En cells, except when they abut Odd cells (Fig. 4F-F''), supporting our hypothesis that both early specification of Odd cells, as well as their subsequent maintenance, is relevant to groove development. Wg therefore patterns the segment boundary by maintaining the En identity among cells within the reach of the Wg signal, while cells beyond the reach of Wg adopt the groove identity and become part of the next segment.
Wg does not repress groove identity once Odd expression is established
We next tested whether Wg controls only the choice between maintaining En
and adopting the groove identity or whether it can also repress groove
differentiation at later stages as proposed previously
(Larsen et al., 2003;
Piepenburg et al., 2000). To
do this, we analyzed Pnr-Gal4; UAS Wg embryos, in which Wg is
ectopically expressed in the dorsal epidermis. Pnr-Gal4 drives
expression beginning at stage 10-11 (after tracheal invagination and before
groove morphogenesis), until late embryonic stages. Ectopic Wg is therefore
delivered after the En/Odd transition has occurred and includes the full
period of groove morphogenesis. The ectopic expression is similar in strength
to the endogenous Wg expression in the ventral epidermis
(Fig. 4I). These embryos show
no sign of Odd weakening, and the grooves are not affected
(Fig. 4I',I'').
Therefore, Wg does not repress Odd expression or groove identity at later
stages, indicating that Wg blocks the specification of the Odd-expressing
groove precursor cells during a specific time window. As we demonstrate below,
this mechanism is distinct from the mechanism that later refines the
population of Odd-expressing groove precursors.
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;
Taylor et al., 1993), and is
required for groove formation (Larsen et
al., 2003), we investigated whether it controls this refinement of
Odd expression. In hh null mutants, Odd expression is normal or
slightly wider than wild type until stage 10
(Fig. 5A,A'), and the
dorsal and ventral Odd populations are present on either side of the tracheal
placode. At stage 11, Odd expression decreases compared with controls, and
disappears by stage 12 (Fig.
5B,B'). When the germ band retracts, no Odd is detected in
the epidermis and no grooves form. The effects of too much Hh activity were
first assayed in patched (ptc) mutants; Ptc is the Hh
receptor and functions as an antagonist of signaling
(Chen and Struhl, 1996;
Ingham et al., 1991). In
ptc mutants, in which Hh signaling is activated in all non-En cells,
Odd is expressed at stage 12 in stripes broader than wild-type stripes
(Fig. 5C,C'). The
refinement step does not occur, and from stage 13 through stage 15, broad
stripes of Odd persist. A similar result was obtained when Hh was
overexpressed in the En domain (Fig.
5D,D'). Thus, while Hh absence causes an excessive reduction
of the Odd-expressing population, Hh excess prevents the refinement of the Odd
population, instead resulting in the maintenance of what appears to be the
full population of Odd cells previously established when Wg signaling patterns
the width of the En stripes. We next asked whether the additional Odd-expressing cells, present when Hh signaling is inappropriately maintained, differentiate into groove cells. In ptc mutants, broad Odd domains are maintained, in which all cells express Crb, aPKC and Ena in the characteristic groove manner and display smaller diameters (Fig. 5G,G'). All the groove cells of a given segment form a single wide groove (Fig. 5G''). Edges of the grooves form regardless of the presence or absence of ectopic En cells that are characteristic of Ptc mutants. Similar wide grooves form when Hh is overexpressed, a situation in which ectopic En cells are never present, ruling out a requirement for direct contact with En cells for groove formation.
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), indicating that they are responding to Hh signal
(Fig. 5E-E''), while
Ptc expression decreases to its basal level in the cells that lose
Odd (Fig. 5F-F'''). The
synchronous reduction of Ptc and Odd expression, which correspond,
respectively, to the pathway activation and the biological output, shows that
this system adapts very quickly to the withdrawal of Hh. Our data show that Hh directs the refinement of a population of groove cells to a single row of cells. If Hh signaling is disrupted, or if it is expanded, either too few or too many cells maintain the groove identity, resulting respectively in the subsequent absence or the widening of the grooves. This mechanism is independent of, and occurs after the specification of the groove identity controlled by early Wg.
Hh does not induce groove identity
Groove cells are not induced by Hh in non-Odd expressing territories, such
as the dorsal and ventral Wg-expressing cells or the lateral cells on the
anterior side of the En stripes. In the ventral domain, Hh does not induce
groove cells anterior or posterior to the En cells. To rule out that Hh could
initiate groove identity if non-Odd cells were present immediately posterior
to En cells in the lateral domain, we genetically displaced cells. Activated
Yan (Rebay and Rubin, 1995)
overexpression compromises the integrity of the En stripe, allowing some cells
to cross the segmental boundary and intercalate between the converging Odd
populations during tracheal invagination
(Fig. 6A,A'). These cells
lose En expression, become receptive to Hh and express Ptc at levels similar
to their dorsal and ventral neighbors (Fig.
6B-B''). Still, they do not express Odd or adopt a groove
identity (Fig. 6C-C'') but
instead interrupt the groove. As a control, ectopic expression of activated
Yan in Odd cells before or after groove differentiation does not interfere
with groove formation (data not shown). As the En cells are the nearest
lineage to the Odd cells, this is the most stringent possible test of the
ability of Hh to induce groove in non-Odd cells. This strongly suggests that
Hh signaling cannot induce groove identity in non-Odd cells.
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DISCUSSION |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Martinez-Arias et al., 1988).
Our demonstration that Wg patterns the En cells, although it does not specify
them, is consistent with the notion that specification and patterning can be
regulated by independent mechanisms
(Martinez-Arias and Hayward,
2006).
How might Wg signal the choice to maintain En identity rather than switch
to the Odd-expressing identity? A previous characterization of the En enhancer
(Florence et al., 1997) may
help understand the molecular mechanism governing the En/Odd identity switch.
The En enhancer contains Ftz Ftz-F1-binding sites sufficient to establish
reporter expression (see Fig. S5 in the supplementary material). A Ftz Ftz-F1
reporter is expressed in a wide domain and is not refined to a two cell wide
stripe as is wild-type En, showing that its expression is maintained
independently of Wg. A specific region of the En enhancer is needed to refine
expression, and was hypothesized to contain Odd-binding sites, as Odd limits
En expression (Coulter and Wieschaus,
1988). Wg signaling acts through the TCF transcription factor, and
TCF-binding sites are located between the Ftz Ftz-F1-binding sites and the
repressing region. The TCF sites do not modify expression of the Ftz Ftz-F1
reporter, but do limit refinement to the normal width stripe when the proposed
Odd-binding sequences are present. In the wild-type enhancer, we propose that,
through the TCF-binding sites, Wg signaling maintains expression by preventing
Odd-mediated repression. Consistent with this interpretation, ectopic Wg
expression after the switch from En to Odd expression but before grooves
differentiate shows that Wg cannot repress either Odd expression or groove
morphogenesis (Fig.
4I',I''). This might therefore provide a molecular
model for the ability of Wg to block a switch in cell identity.
Following En patterning, the remaining Odd cells posterior to the En domain are again partitioned into two domains: the anterior domain retaining Odd and the posterior domain losing Odd expression. Our data show that Hh, which is produced by the En cells, maintains Odd expression within its reach, which in wild type is progressively narrowed to a single row adjacent to the En domain. The Odd cells differentiate into grooves, whereas the cells that lose Odd produce a characteristic cuticular hair at later stages. The close temporal relationship between reduction of Ptc expression and loss of Odd expression suggests a relatively close coupling between Hh signal and Odd expression.
Because Odd expression is associated with the groove cell identity, the maintenance of Odd expression in some of the Odd-expressing cells suggests that Hh maintains the groove identity in cells within reach of the signal. However, because we do not know how to maintain the Odd-associated identity in the absence of Hh signaling, we cannot discern whether Hh is also required to provide a signal in addition to that needed to maintain the groove identity. Whether or not such a hypothetical additional signal might be required, the direct correspondence between Odd maintenance and groove differentiation demonstrates that this hypothetical signal would not provide any information beyond that associated with the binary decision to maintain the Odd/groove cell identity, and would therefore not be instructive in the usual sense of specifying between alternative developmental pathways.
The cell types posterior to the En domain have previously been studied
based on the type of cuticle they secrete. The groove cells secrete naked
cuticle, whereas their posterior neighbors secrete thick pigmented hairs.
Using these markers of cell identity, it was proposed that Hh acts as a
morphogen, inducing at high concentration the fate of naked cuticle-producing
cells, and at lower concentration the fate of thick pigmented hair-secreting
cells (Heemskerk and DiNardo,
1994). By monitoring Ptc and Odd expression in the wild-type
embryo, we observe elevated levels in the Odd (groove) domain, and basal
levels as soon as Hh signaling is withdrawn and the groove identity is
abandoned in favor of the thick pigmented hair identity. Our data are
therefore more consistent with the interpretation that Hh exceeds the
threshold for signaling in the naked cuticle (Odd) domain, but drops below
threshold in the thick pigmented hair-secreting domain. Heemskerk and DiNardo
also observed an expansion of the thick pigmented hair domain and a decrease
in the posterior fine hair domain with elevated Hh levels
(Heemskerk and DiNardo, 1994).
Because in wild type we observe equal, basal, Ptc expression in this region,
we propose that this boundary is not normally patterned by Hh. We hypothesize
that with elevated Hh, decreased cell death in the thick pigmented hair domain
and competition with the posterior fine hair domain produce this altered
pattern. A single Hh threshold would be incompatible with the morphogen
model.
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; Jones et al.,
1995; Vincent et al.,
1996). In the absence of Wg signaling, dorsal epidermal cells
switch from En to groove identity and, subsequently, if Hh is lost, from
groove to a third, thick hair identity. It is, as yet, unknown whether these
identity switches depend on exogenous signals, or whether they are cell
autonomously controlled. Furthermore, how their timing is regulated remains
unknown. Wg acts during a specific time window, implying that the patterning
signal must persist as long as the transcription factors responsible for the
switch are active. The patterning signal has no effect on the pace of the
progression. Patterning of the dorsal epidermis is controlled by the
persistence and reach of the signal within the time window, when the switch is
functional, and not by the concentration of the signal
(Fig. 7). Our observations suggest that cell identity and differentiation are independently controlled. Groove differentiation begins before refinement of the Odd domain is complete, as the two remaining rows of Odd cells begin to express some groove differentiation markers (see Fig. S1 in the supplementary material), showing that differentiation depends on the identity of the cell at that time. As the Odd domain is further narrowed to a single cell stripe, differentiation markers are lost along with Odd expression from the row further from the Hh/En cells. Because grooves develop specifically from cells that express the Odd marker well before groove morphogenesis, and because signaling to maintain Odd precedes morphogenesis, we conclude that morphogenesis requires cell prior competence or specification, and does not result simply from the direct influence of signaling on cellular morphogenesis.
How general is this inhibition of cell identity progression by patterning
signals? This mechanism may be relevant to the development of stem cells, the
environment of which provides stabilizing signals that maintain stemness. For
example, vertebrate homologues of Wg (Wnt), have been proposed to maintain
stemness in hematopoietic stem cells (HSCs)
(Reya et al., 2003;
Willert et al., 2003). It is
likely that Wnt signaling blocks the transition from HSC to the multipotent
progenitor identity. Importantly, progenitors may need to escape the Wnt
signal in order to proceed through hematopoiesis, as transient Wnt stimulation
enhances hematopoietic reconstitution, but constitutive signaling induced by
transgenic pathway activation prevents the appearance of progenitors, and
hematopoiesis fails (Kirstetter et al.,
2006; Scheller et al.,
2006). It is unclear why similar experiments using retroviral
signal activation produced different results
(Reya et al., 2003).
Later stages of hematopoiesis appear to be similarly regulated by Wnt
signaling. In vivo constitutive activation of the Wnt pathway blocks
development of multiple hematopoietic lineages without affecting already
differentiated cells (Kirstetter et al.,
2006; Scheller et al.,
2006). Transitions between blood cell identities may therefore
constitute steps controlled by Wg signaling, analogous to the switch we
describe between En and Odd identities. Additional work will be needed to test
whether hematopoietic stem cell development is controlled by signal-dependent
antagonism of switches governing a progression of cell identity changes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/16/2767/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Alexandre, C., Lecourtois, M. and Vincent, J.
(1999). Wingless and Hedgehog pattern Drosophila denticle belts
by regulating the production of short-range signals.
Development 126,5689
-5698.[Abstract]
Baker, N. E. (1987). Molecular cloning of
sequences from wingless, a segment polarity gene in
Drosophila: The spatial distribution of a transcript in embryos.
EMBO J. 6,1765
-1773.[Medline]
Cadigan, K. M., Grossniklaus, U. and Gehring, W. J.
(1994). Localized expression of sloppy paired protein maintains
the polarity of Drosophila parasegments. Genes Dev.
8, 899-913.
Chen, Y. and Struhl, G. (1996). Dual roles for
patched in sequestering and transducing Hedgehog. Cell
87,553
-563.[CrossRef][Medline]
Coulter, D. E. and Wieschaus, E. (1988). Gene
activities and segmental patterning in Drosophila: analysis of odd-skipped and
pair-rule double mutants. Genes Dev.
2,1812
-1823.
Coulter, D. E., Swaykus, E. A., Beran-Koehn, M. A., Goldberg,
D., Wieschaus, E. and Schedl, P. (1990). Molecular analysis
of odd-skipped, a zinc finger encoding segmentation gene with a novel
pair-rule expression pattern. EMBO J.
9,3795
-3804.[Medline]
DiNardo, S. and O'Farell, P. H. (1987).
Establishment and refinement of segmental pattern in the Drosophila embryo:
spatial control of engrailed expression by pair rule genes.
Genes Dev. 1,1221
-1225.
DiNardo, S., Sher, E., Heemskerk-Jongens, J., Kassis, J. A. and
O'Farrell, P. H. (1988). Two-tiered regulation of spatially
patterned engrailed gene expression during Drosophila
embryogenesis. Nature
332,604
-609.[CrossRef][Medline]
Fjose, A., McGinnis, W. J. and Gehring, W. J.
(1985). Isolation of a homeobox-containing gene from the
engrailed region of Drosophila and the spatial distribution
of its transcript. Nature
313,284
-289.[CrossRef][Medline]
Florence, B., Guichet, A., Ephrussi, A. and Laughon, A.
(1997). Ftz-F1 is a cofactor in Ftz activation of the Drosophila
engrailed gene. Development
124,839
-847.[Abstract]
Forbes, A. J., Nakano, Y., Taylor, A. M. and Ingham, P. W.
(1993). Genetic analysis of hedgehog signalling in the Drosophila
embryo. Development Suppl.115
-124.
Grevengoed, E. E., Loureiro, J. J., Jesse, T. L. and Peifer,
M. (2001). Abelson kinase regulates epithelial morphogenesis
in Drosophila. J. Cell Biol.
155,1185
-1198.
Hatini, V., Green, R. B., Lengyel, J. A., Bray, S. J. and
Dinardo, S. (2005). The Drumstick/Lines/Bowl regulatory
pathway links antagonistic Hedgehog and Wingless signaling inputs to epidermal
cell differentiation. Genes Dev.
19,709
-718.
Heemskerk, J. and DiNardo, S. (1994).
Drosophila hedgehog acts as a morphogen in cellular patterning.
Cell 76,449
-460.[CrossRef][Medline]
Hosoya, T., Takizawa, K., Nitta, K. and Hotta, Y.
(1995). glial cells missing: a binary switch between neuronal and
glial determination in Drosophila. Cell
82,1025
-1036.[CrossRef][Medline]
Ingham, P., Taylor, A. and Nakano, Y. (1991).
Role of the Drosophila patched gene in positional signaling.
Nature 353,184
-187.[CrossRef][Medline]
Jones, B. W., Fetter, R. D., Tear, G. and Goodman, C. S.
(1995). glial cells missing: a genetic switch that controls glial
versus neuronal fate. Cell
82,1013
-1023.[CrossRef][Medline]
Kirstetter, P., Anderson, K., Porse, B. T., Jacobsen, S. E. and
Nerlov, C. (2006). Activation of the canonical Wnt pathway
leads to loss of hematopoietic stem cell repopulation and multilineage
differentiation block. Nat. Immunol.
7,1048
-1056.[CrossRef][Medline]
Kornberg, T., Siden, I., O'Farrell, P. and Simon, M.
(1985). The engrailed locus of Drosophila: in situ localization
of transcripts reveals compartment-specific expression.
Cell 40,45
-53.[CrossRef][Medline]
Larsen, C. W., Hirst, E., Alexandre, C. and Vincent, J. P.
(2003). Segment boundary formation in Drosophila embryos.
Development 130,5625
-5635.
Lawrence, P. A., Johnston, P., Macdonald, P. and Struhl, G.
(1987). Borders of parasegments in Drosophila embryos are
delimited by the fushi tarazu and even-skipped genes.
Nature 328,440
-442.[CrossRef][Medline]
Macdonald, P. M., Ingham, P. and Struhl, G.
(1986). Isolation, structure, and expression of even-skipped: a
second pair-rule gene of Drosophila containing a homeo box.
Cell 47,721
-734.[CrossRef][Medline]
Martinez-Arias, A. (1993). Development and
patterning of the Drosophila epidermis. In The Development
of Drosophila melanogaster, Vol. 1 (ed. M.
Bate and A. Martinez-Arias), pp. 517-608. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory Press.
Martinez-Arias, A. and Lawrence, P. A. (1985).
Parasegments and compartments in the Drosophila embryo.
Nature 313,639
-642.[CrossRef][Medline]
Martinez-Arias, A. and Hayward, P. (2006).
Filtering transcriptional noise during development. Nat. Rev.
Genet. 7,34
-44.[CrossRef][Medline]
Martinez-Arias, A., Baker, N. and Ingham, P. W.
(1988). Role of segment polarity genes in the definition and
maintenance of cell states in the Drosophila embryo.
Development 103,153
-170.
Nusslein-Volhard, C. and Wieschaus, E. (1980).
Mutations affecting segment number and polarity in Drosophila.
Nature 287,795
-801.[CrossRef][Medline]
Oda, H. and Tsukita, S. (1999). Dynamic
features of adherens junctions during Drosophila embryonic epithelial
morphogenesis revealed by a Dalpha-catenin-GFP fusion protein. Dev.
Genes Evol. 209,218
-225.[CrossRef][Medline]
Perrimon, N., Engstrom, L. and Mahowald, A. P.
(1989). Zygotic lethals with specific maternal effect phenotypes
in Drosophila melanogaster. I. Loci on the X chromosome.
Genetics 121,333
-352.
Perrimon, N., Lanjuin, A., Arnold, C. and Noll, E.
(1996). Zygotic lethal mutations with maternal effect phenotypes
in Drosophila melanogaster. II. Loci on the second and third chromosomes
identified by P-element-induced mutations. Genetics
144,1681
-1692.[Abstract]
Piepenburg, O., Vorbruggen, G. and Jackle, H.
(2000). Drosophila segment borders result from unilateral
repression of hedgehog activity by wingless signaling. Mol.
Cell 6,203
-209.[CrossRef][Medline]
Rebay, I. and Rubin, G. M. (1995). Yan
functions as a general inhibitor of differentiation and is negatively
regulated by activation of the Ras1/MAPK pathway. Cell
81,857
-866.[CrossRef][Medline]
Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C.,
Willert, K., Hintz, L., Nusse, R. and Weissman, I. L. (2003).
A role for Wnt signalling in self-renewal of haematopoietic stem cells.
Nature 423,409
-414.[CrossRef][Medline]
Saulier-Le Drean, B., Nasiadka, A., Dong, J. and Krause, H.
M. (1998). Dynamic changes in the functions of Odd-skipped
during early Drosophila embryogenesis. Development
125,4851
-4861.[Abstract]
Scheller, M., Huelsken, J., Rosenbauer, F., Taketo, M. M.,
Birchmeier, W., Tenen, D. G. and Leutz, A. (2006).
Hematopoietic stem cell and multilineage defects generated by constitutive
beta-catenin activation. Nat. Immunol.
7,1037
-1047.[CrossRef][Medline]
Spemann, H. and Mangold, H. (1924). Über
induktion von Embryonalagen durch Implantation Artfremder Organisatoren.
Roux' Arch. Entw. Mech. Org.
100,599
-638.
Tabata, T. and Kornberg, T. B. (1994). Hedgehog
is a signaling protein with a key role in patterning Drosophila imaginla
discs. Cell 76,89
-102.[CrossRef][Medline]
Taylor, A. M., Nakano, Y., Mohler, J. and Ingham, P. W.
(1993). Contrasting distributions of patched and hedgehog
proteins in the Drosophila embryo. Mech. Dev.
42, 89-96.[CrossRef][Medline]
Tepass, U., Theres, C. and Knust, E. (1990).
crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila
epithelial cells and required for organization of epithelia.
Cell 61,787
-799.[CrossRef][Medline]
van den Heuvel, M., Klingensmith, J., Perrimon, N. and Nusse,
R. (1993). Cell patterning in the Drosophila segment:
engrailed and wingless antigen distributions in segment polarity mutant
embryos. Development Suppl.105
-114.
Vincent, S., Vonesch, J. L. and Giangrande, A.
(1996). Glide directs glial fate commitment and cell fate switch
between neurones and glia. Development
122,131
-139.[Abstract]
Willert, K., Brown, J. D., Danenberg, E., Duncan, A. W.,
Weissman, I. L., Reya, T., Yates, J. R., 3rd. and Nusse, R.
(2003). Wnt proteins are lipid-modified and can act as stem cell
growth factors. Nature
423,448
-452.[CrossRef][Medline]
Wodarz, A., Ramrath, A., Grimm, A. and Knust, E.
(2000). Drosophila atypical protein kinase C associates with
Bazooka and controls polarity of epithelia and neuroblasts. J. Cell
Biol. 150,1361
-1374.
Zeng, W., Wharton, K. A., Jr, Mack, J. A., Wang, K., Gadbaw, M.,
Suyama, K., Klein, P. S. and Scott, M. P. (2000). naked
cuticle encodes an inducible antagonist of Wnt signalling.
Nature 403,789
-795.[CrossRef][Medline]
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500 µm long and cell diameters after
stage 11 are 
