Notch and tracheal development
The Drosophila tracheal system consists of a stereotyped
network of epithelial tubes formed by several tracheal cell
types. By the end of embryogenesis, when the general
branching pattern is established, some specialized tracheal
cells then mediate branch fusion, while others extend fine
terminal branches. Evidence is presented that the
Notch signaling pathway acts directly in the tracheal cells
to distinguish individual fates within groups of equivalent
cells. Notch helps to single out those tracheal cells that
mediate branch fusion by blocking their neighbours from
adopting the same fate. This function of Notch would
require the restricted activation of the pathway in specific
cells. In addition, and probably later, Notch also acts in the
selection of those tracheal cells that extend the terminal
branches. Both the localized expression and the mutant
phenotypes of Delta, coding for a known ligand for Notch, suggest that
Delta may activate Notch to specify cell fates at the tips of
the developing tracheal branches (Llimargas, 1999).
Zygotic N mutant embryos show abnormalities in most of
the ectodermal derivatives, including the tracheal system,
where some of the tracheal cells are converted into neuroblasts; the embryos exhibit only
rudimentary branches, most probably due to the loss of tracheal
cells. In addition, abnormalities in fusion and
terminal branching are also detected when luminal or cellular
markers are used.
These phenotypes could be due to early
requirements for the N pathway before tracheal fate
is allocated. To detect late N requirements, Nts
mutant embryos (Nts over a null N allele) were
shifted to the restrictive temperature at stage 11. The
terminal branching and fusion phenotypes produced
resemble those observed in zygotic null N mutants,
yet the defects in the primary branching pattern are
milder. The phenotypes observed both in
Nts combinations and in null N mutants suggest that,
in addition to its early role in tracheal specification,
N acts later in both fusion and terminal branching
programs (Llimargas, 1999).
To analyse the requirements of N in fusion and terminal
branching, the identity of the tracheal cells was studied using
the expression of several tracheal markers. In wild-type
embryos, tracheal cells that mediate branch fusion express the
fusion markers; a single fusion cell is found at the tips of the
following branches: DB, lateral trunk anterior (LTa),
lateral trunk posterior (LTp), dorsal trunk anterior (DTa) and
dorsal trunk posterior (DTp). Conversely, the antifusion
markers are generally expressed in all other tracheal cells. Finally, the terminal markers are expressed in about 20
cells per tracheal metamere. Those are the cells, either
clustered or isolated, that will extend terminal branches from
most primary branches except the DT (Llimargas, 1999).
Zygotic null mutants of N show abnormal patterns for these
molecular markers. In the mutants, Fusion-1 (escargot), Fusion-2 and Fusion-3
(two unidentified enhancer traps used as tracheal markers) are
ectopically expressed in almost all the cells forming the
rudimentary DB, DT and LT. The expression of
Terminal-1 (pruned) is found mainly in
some cells along the transverse connective (TC) and the
visceral branches (VB), but almost never in the DB. Antifusion-1 and Antifusion-2 (two
unidentified tracheal markers) are consistently expressed only
in the TC.
Similarly, the overexpression of Hairless (H), a Notch signaling antagonist, in the tracheal cells also
causes changes in the expression of these tracheal markers. In
most embryos, there is an increase in the number of cells
expressing fusion markers: DBs contain up to four cells
expressing Fusion-1 and Fusion-2 as compared to
one cell in the wild type; and LT and DT contain up to four fusion
cells, as compared to two cells in normal conditions. Conversely,
there is a decrease in the number of cells expressing Terminal-1,
mainly in the DBs and in the GBs, which in
wild-type embryos contain a single cell expressing the terminal
markers. The pattern of expression of Antifusion-1 is similar
to wild type, although there is one fewer cell on average in
DBs expressing the marker. Cell counts in the DBs
of wild type and of embryos overexpressing H indicate that
there is no change in the number of cells when N function is
reduced. This suggests
that the extra fusion cells observed may arise by a
transformation of presumptive terminal or antifusion cells into
fusion cells.
The formation of ectopic terminal branches observed
sporadically when overexpressing H correlates with the
expression of Terminal-1 in more than one cell in some DBs and GBs. The presence of extra terminal branches
suggests a second N role in selecting the correct number of
terminal cells. Engineered forms of the N protein that eliminate the
extracellular domain are active in the absence of ligand. If N is required for the
execution of terminal branching and fusion programs, a
constitutively activated form of N could cause a transformation that is the opposite
of the loss of N function, and indeed this was found to be true (Llimargas, 1999).
Cells expressing fusion and terminal markers arise from the
tips of the primary branches. At early stages of tracheal
morphogenesis, broad domains at the tips of each budding
primary branch express the pantip markers. Their pattern of expression suggests that the tip cells
are apparently equivalent before adopting individual fates.
The N pathway affects the early
diversification of the tip cells. pointed (pnt, Pantip-1) and sprouty (spry, Pantip-2) are expressed in each DB in about 3 to 4 cells at
stages 12 and 13; at stage 13/14, high levels are found in the
two tip cells and at later stages expression is restricted to
the cell expressing the terminal markers. Under N loss-of-function
conditions, the expression of pnt and spry in the
DBs is frequently lost from stage 13 while under N gain-of-function
conditions high levels of pnt and spry are maintained
in more than one cell at stage 16. This suggests that the activity of
N allows the tip cells to diversify. This also suggests a possible
repression of these pantip markers by the fusion genes, since their
expression decays in the cells expressing the fusion factors (Llimargas, 1999).
How is the activity of N regulated in the tracheal system?
Dl has been shown to act as a ligand for N. Zygotic
null N mutants have a weaker tracheal phenotype than that of
null Dl mutants, possibly because the maternal N product
rescues the zygotically mutant embryos.
The tracheal branches in Dl mutants are so truncated that it is
not possible to determine effects on fusion or terminal
branching. The overexpression of Dl driven by the
breathless-Gal4 line affects fusion and terminal branching in ways
similar to both the gain and loss of N function.
Thus, ectopic fusions and unfused branches are observed with variable
frequency, as
well as missing or extra terminal branches. These phenotypes correlate with both the lack and the excess
of cells expressing fusion and terminal markers.
The expression pattern of Dl in the tracheal cells was
studied in detail using both in situ hybridization and antibody
staining to further clarify Dl function. The Dl protein
accumulates in vesicles at higher levels in the tip cells of the
primary branches from stage 12 to 13. The localized
accumulation of the protein is coextensive with a localized
expression of the gene in the tip cells. At later stages,
large amounts of transcripts and protein are found in the DT.
The early accumulation of Dl depends on N activity. Dl
accumulates at low levels at the tips of the branches in embryos
with constitutively active N, while in null N mutants, there is a broader
accumulation of Dl protein.
The coincidence of the early Dl expression with the activity
of the N pathway and the phenotypes of Dl overexpression,
suggests that Dl activates N to diversify the tip cells of the
primary branches (Llimargas, 1999).
A model is presented for the establishment of tracheal fates at
the tips of the branches.
bnl, coding for the FGF ligand, is expressed in clusters of cells outside the tracheal system
and, in addition to guiding branch migration, it is necessary to
pattern the tips of the primary branches. Bnl activates the Breathless receptor in a gradient, leading to
the expression of the pantip, terminal and fusion markers. Two pieces of evidence indicate that
the tip cells are initially equivalent: (1) they all express the
pantip markers in response to bnl and, (2) they can all
behave in the same way in different mutant backgrounds. The
expression of the pantip markers becomes restricted to those
cells at the leading ends of the branches, since the Breathless (Btl) activity is
higher there due to the proximity of the Bnl source. Among these
tip cells, some become fusion cells while others can
differentiate as terminal cells, even though they all receive
similar amounts of Bnl. Therefore, the bnl/btl pathway is not
sufficient to account for the diversification of the tip cells, and
it is proposed that the N signaling acts to achieve this.
The pnt gene (Pantip-1) has been shown to repress the fusion
markers, yet even though the tip
cells all express pnt, only one of them acquires the fusion fate. A possible resolution of this paradox might be that there is
a balance between the bnl/btl pathway, which promotes the
fusion fate, and the N signal and pnt, which represses it. The N
pathway shifts this balance: its presumed inactivity in one cell
would allow that cell to overcome the repression by pnt, while
its activity in the remaining cells would allow them to
overcome the activation by bnl/btl. It is possible that biasing
differences in the tip cells are also required to shift the balance.
Once the fusion cell is specified, the expression of some
pantip markers decays in that cell, suggesting that the fusion
factors repress the pantip genes. A piece of evidence supports
this: in N mutants, where all the cells express the fusion
markers, Pantip-1 and Pantip-2 expressions are extinguished.
Simultaneously, the fusion cell seems to signal via Dl to its
neighbors to repress the fusion genes, allowing them to
express the antifusion or the terminal genes. The data suggest
that the fusion genes repress the antifusion ones, because in
the wild type the antifusion and the fusion genes are expressed
in complementary patterns and, in null N mutants (where all
the cells express the fusion markers), the antifusion genes are
repressed.
Similarly, the specification of the terminal fate also depends
on a balance between inductive signals, mediated by bnl/btl
and the pantip genes, and repressive signals, mediated by N,
spry and hdc. Cells closest to the Bnl source that also
express high levels of pantip markers and that do not acquire
the fusion fate, can become terminal cells. The remaining cells
receive negative signals from the already specified terminal cell
(mediated by Dl and spry) and from the also specified fusion
cell (mediated by the novel protein Headcase, the Fusion 6 marker) to repress the terminal fate. Thus, the
acquisition of fates among a group of cells located at the tips
of each primary branch depends on the integration of positive
and negative effects from different signaling pathways (Llimargas, 1999 and references).
The patterned branching in the Drosophila tracheal system
is triggered by the FGF-like ligand Branchless (Bnl) that
activates a receptor tyrosine kinase Breathless (Btl) and the
MAP kinase pathway. A single fusion cell at the tip of each
fusion branch expresses the zinc-finger gene escargot, leads
branch migration in a stereotypical pattern and contacts
with another fusion cell to mediate fusion of the branches.
A high level of MAP kinase activation is also limited to the
tips of the branches. Restriction of such cell specialization
events to the tip is essential for tracheal tubulogenesis. Notch signaling plays crucial roles in the
singling out process of the fusion cell. Notch
is activated in tracheal cells by Branchless signaling
through stimulation of Delta (Dl) expression at the tips of
tracheal branches and activated Notch represses the
fate of the fusion cell. In addition, Notch is required to
restrict activation of MAP kinase to the tips of the branches,
in part through the negative regulation of Branchless
expression. Notch-mediated lateral inhibition in sending
and receiving cells is thus essential to restrict the inductive
influence of Branchless on the tracheal tubulogenesis (Ikeya, 1999).
Six primary branches form in the tracheal primordia,
among which the dorsal branch (DB), anterior and posterior
dorsal trunk (DTa, DTp), and anterior and posterior lateral
trunk (LTa, LTp) migrate along a stereotyped path to be
connected with other branches from adjacent primordia. These fusion branches are capped with fusion cells that
express Esg. The remaining visceral
branch (VB) migrates to reach the internal organs. Terminal
cells expressing Drosophila serum response factor (DSRF) are formed in each primary branch except in DTs and
later differentiate multiple tracheoles.
High expression of DL mRNA and
protein is expressed in the DT of stage-15 embryos. Cells in the tracheal primordium just
after invagination expressed Dl uniformly. At
early stage 11, Dl expression started to be elevated in 2-3 cells
at the tip of the branches in which outgrowth had begun and the number of the Dl-expressing cells was reduced to
one at late stage 11. At stage 14, high Dl expression
remains only in the DT, which has completed fusion. Ser protein also accumulates at the
apical side of the DT cells at the same stage. In the case of the trachea, the level of N protein
expression remains uniform, suggesting that the
expression level of Dl, or its potentiation, must be crucial for
N activation. An esg-lacZ reporter is initially
expressed in 2-3 cells at the tip of the fusion branches in mid
stage 11 embryos, and is downregulated to
be maintained in only a single cell at the tip of each fusion
branch at late stage 11. These cells also express
a high level of Dl. Therefore, localized elevation
of Dl expression in stage 11 correlates well with the selection
process of a single fusion competent cell (Ikeya, 1999).
When
tracheal cells became unresponsive to Bnl due to btl
mutation, no sign of primary
branching and Dl upregulation is observed. On
the contrary, when Btl is hyperactivated by overexpression
of Bnl in all the tracheal cells, primary branching is severely
inhibited and Dl expression is elevated. These
results suggest that elevation of Dl expression is triggered by
the external signal Bnl. The results also suggest that the N/Dl
pathway may mediate the Bnl signal to control cell migration
and cell fate decisions (Ikeya, 1999).
In Nts1 embryos grown under non-permissive condiditons a misrouting defect was observed in DB, which
normally elongates to the dorsal midline where it meets its
counterpart from the other side of the metamere.
DBs are often curved in the anteroposterior
direction and make contact with the tip of DB from the same
side. The misrouted DBs accumulate a luminal
component detectable by 2A12 antibody at the ectopic contact
sites, but do not appear to fuse properly. Cell migration defect is also observed in DB. DB consists of
a total of 5-7 cells in the case of Tr5, of which two specialized cells are located at the
tip. One is the terminal cell, from
which a thin terminal branch sprouts: the other is the fusion
cell. The remaining stalk cells are located between the tip and DT at regular
intervals. In Nts1 mutants, the number of cells at the tip is
increased with a corresponding decrease in
the number of stalk cells, the latter having become unusually
elongated. The total number of cell nuclei do
not change compared to the controls, so no additional mitosis
occurs. This cell migration phenotype suggests that
stalk cells and terminal cells acquire a property of fusion cells
to become localized at the tip of DB. Since Esg represses DSRF
expression and terminal branching,
the loss of terminal branching in Nts1 embryos may be the
consequence of ectopic Esg expression. Similar defects in Esg
and DSRF expression are also observed in null
N mutants. These results suggest that in N minus
embryos, several terminal and stalk cells are recruited to the
fate of fusion cells (Ikeya, 1999).
A three-step model is presented for tubule formation in fusion branches.
During induction, exogenously supplied Bnl activates its receptor Btl in
equivalent tracheal cells where N is inactive. The signal is
transduced by activation of MAPK to
stimulate Dl expression. The expression of Bnl is also regulated
negatively by N signaling. During lateral inhibition, induced Dl
activates N in neighboring cells, which in turn, represses esg
transcription. N may also repress Dl expression. However, a high
level of Dl inhibits N signaling in a cell autonomous manner,
allowing activation of esg and MAPK. A small difference in the
response to Bnl within equivalent tracheal cells is amplified to select
out a single fusion cell with a high level of Dl and esg expression.
During tubule formation, the fusion cell becomes the only cell that
responds to Bnl and becomes motile. Maintenance of Btl activity by
Bnl would limit the migration toward the source of Bnl. Other
tracheal cells follow fusion cells to become stalk cells (Ikeya, 1999).
Decapentaplegic (Dpp) signaling determines the number of cells that
migrate dorsally to form the dorsal primary branch during tracheal
development. Dpp is expressed in dorsolateral epidermal clusters located
near the tips of the outgrowing dorsal branches. The Dpp receptor, Thick
veins, is expressed in all tracheal cells during embryogenesis and is required
for in dorsal branch outgrowth ectopic activation of fusion markers in cells
of the dorsal branch. Dpp signaling is required for the differentiation of one
of three different cell types in the dorsal branches, the fusion cell. In
Mad mutant embryos or in embryos expressing dominant negative
constructs of the two type I Dpp receptors in the trachea the number of cells
expressing fusion cell-specific marker genes is reduced and fusion of the
dorsal branches is defective. Ectopic expression of Dpp or the activated form
of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the
tracheal lumen and ectopic expression of fusion gene markers in all tracheal
branches. Delta is among the fusion marker genes that are activated in the trachea
in response to ectopic Dpp signaling. In conditional
Notch loss of function mutants, additional tracheal cells adopt the
fusion cell fate. Ectopic expression of an activated form of the Notch
receptor in fusion cells results in suppression of fusion cell markers and
disruption of the branch fusion. The number of cells that express the fusion
cell markers in response to ectopic Dpp signaling is increased in Notch
ts1 mutants, suggesting that the two signaling pathways have opposing
effects in the selection of the fusion cells in the dorsal branches (Steneberg,
1999).
Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Genetic mosaic analysis has been used in Drosophila to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them (Ghabrial, 2006).
Because small differences in btl dosage or activity affect a cell's ability to compete for the lead, whether lateral inhibitory mechanisms that amplify small differences in signalling might be operative was investigated. Data suggest that the Notch pathway, a lateral signalling pathway implicated in cell specification events including cell fate determination at tracheal branch tips, also affects cell arrangement. Nts embryos shifted to the restrictive temperature during budding formed branches in which most DB cells behaved like lead cells, resulting in large clusters of cells congregated at the lead position, whereas expression of constitutively active NACT throughout the tracheal epithelium had the opposite effect, arresting outgrowth and stalling cells near the base of the branch. It is proposed that Notch-mediated lateral inhibition among tracheal cells prevents extra cells from assuming the lead position (Ghabrial, 2006).
These results provide evidence for social stratification and dynamic social interactions between epithelial cells during branching morphogenesis. First, the results show that budding cells are functionally specialized. A cell at the branch tip requires btl and leads outgrowth towards the Bnl signalling center. Trailing cells do not require btl but nevertheless follow the lead cell towards the Bnl source. Because tracheal cells do not migrate or form tubes in btl-/- animals, trailing cells must receive a secondary signal generated by the lead cell that induces them to migrate and also activates their tubulogenesis program. This could be a secreted molecule or physical stimulus such as pulling or stretching the trailing cells (Ghabrial, 2006).
Second, these roles are not pre-specified. Rather, there is competition between cells such that those with high Btl FGFR activity become lead cells whereas those with less or no btl FGFR activity become trailing cells and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibitory signalling between tracheal cells, and it may also be influenced by positive feedback mechanisms such as increased activation and expression of Btl as cells approach the Bnl source. Third, there may be cooperation between cells, because in a genetically mosaic epithelium, tracheal cells with less Btl activity allow those with more activity to move ahead of them (Ghabrial, 2006).
There may be similar social interactions between budding cells in other branching organs. Studies of other branching processes have identified genes selectively expressed in tip cells of budding branches, and in some cases these cells display morphological specializations indicating that they might actively lead outgrowth. However, because most budding branches contain hundreds or thousands of cells, it is difficult to track and manipulate individual cells to investigate social behaviours like those described here. Recent analyses of chimaeric Ret+/Ret- mouse renal ureteric buds in culture and btl mosaic air sacs reveal that cells lacking these receptor tyrosine kinases are excluded from branch tips, indicating that RTK-dependent interactions similar to those described here might be operative in more complex branching events (Ghabrial, 2006).
The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum.
Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine
also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their
elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell
rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis
of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch
pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This
Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).
At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).
Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).
In addition to expressing Dpp, the li-v domain expresses the Notch ligand Delta (Dl); Dl is also expressed in the anterior of both the rectum and the small intestine. Fringe (Fng), a modulator of Notch signaling, is expressed opposite Dl in the Drosophila wing and eye; in the hindgut, Fng is expressed in li-d and the boundary cell rows, opposite the domain of Dl expression in li-d (Iwaki, 2002).
The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).
Embryos lacking Dl function are extremely deformed and do not always have a recognizable hindgut, indicating that function of Dl early in embryogenesis is required to establish and/or maintain the hindgut. Since Dl encodes a ligand for Notch, embryos lacking the zygotic contribution of Notch were examined. Strikingly, Notch mutant hindguts completely lack both boundary cell rows and rings, revealing that Notch signaling is required to establish the boundary cells. The data demonstrate that formation of the boundary cell rows at the border of Dl expression requires the Notch receptor; however, Fng does not appear to be required for this process (Iwaki, 2002).
To further investigate the required role of Dl in establishing the boundary cells, a dominant-negative form of Dl was expressed throughout the hindgut. bynGal4:UAS-Dl.DN embryos show a complete absence of boundary cell rows and rings; this phenotype closely resembles that seen in Notch loss-of-function embryos. Expression of a dominant negative Notch receptor throughout the hindgut results in a similar absence of boundary cell rows and rings. Furthermore, bynGal4 driven expression of UAS-Hairless, which acts to suppress activity of Su(H) also results in an absence of boundary cells. This last result indicates that the Notch signaling required to establish the boundary cells must act through Su(H). In summary, the above results demonstrate required roles in boundary cell specification of the following Notch pathway components: the ligand Dl, the receptor Notch, and the downstream transcription factor Su(H). It is therefore concluded that the Notch signaling pathway is required for boundary cell induction (Iwaki, 2002).
An intriguing observation, given the demonstrated role of the LIN-12/Notch signaling pathway in generation of left¯right asymmetry in the Caenorhabditis elegans intestine is that a large portion of 455.2Gal4:UAS¯Su(H)VP16 hindguts display a reversal of left¯right looping (Iwaki, 2002).
Ectopic expression experiments, taken together with the loss-of-function experiments, demonstrate that establishment of the boundary cell rows requires the juxtaposition of Dl-expressing and Dl-non-expressing cells and signaling via Notch and Su(H). In addition to Notch and spatially restricted Dl, establishment of the anterior ring requires localized activity of Dpp; the posterior ring requires En/Inv activity (which does not need to be localized) and the localized activity of Dl (Iwaki, 2002).
Since the experiments described in the preceding sections show that both spatially localized En/Inv and a boundary of Dl expression are required to establish the boundary cells, it was asked whether En/Inv might control the boundary of Dl expression. In Df(enE) embryos, Dl is not restricted to li-v, but rather is uniform in the hindgut circumference, indicating that en/inv is required to repress Dl. In the large intestine, uniform expression of En/Inv results in an absence of Dl expression. Expression of En/Inv in li-d is thus both necessary and sufficient to restrict Dl expression to li-d. While it represses Dl throughout the large intestine, ectopic En/Inv does not affect Dl expression in the rectum. Embryos with ectopic En/Inv not only express Dl at the anterior of the rectum, they also form the posterior boundary cell ring. Thus a boundary of Dl-expressing with Dl-non-expressing cells is required not only to establish the boundary cell rows but also likely to establish the posterior ring; the posterior ring also requires En/Inv activity, but this activity does not need to be localized (Iwaki, 2002).
Consistent with observations that En and Inv are repressors with the same targets, the data presented in this study demonstrate that Dl expression in the large intestine is restricted to the li-v domain by the repressive activity of En/Inv in li-d (Iwaki, 2002).
The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).
In summary, three steps in the establishment of the Drosophila hindgut boundary cell rows are similar to steps characterized in other Notch dependent boundary-forming systems. (1) A homeodomain transcription factor (En/Inv in the case of the boundary cells) is expressed on one side of the forming boundary; (2) this transcription factor defines two domains, one which expresses Dl and one which does not; (3) Notch activation in the Dl-non-expressing cells that confront Dl-expressing cells leads to a unique cell fate (Iwaki, 2002).
Drosophila muscles originate from the fusion of two types of
myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To
better understand muscle diversity and morphogenesis, a
large-scale gene expression analysis was performed to identify genes differentially
expressed in FCs and FCMs. Embryos derived from
Toll10b mutants were employed to obtain primarily muscle-forming
mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM
fate, respectively. The transcripts present in embryos of each genotype were
compared by hybridization to cDNA microarrays. Among the 83 genes
differentially expressed, genes known to be enriched in FCs or FCMs,
such as heartless or hibris, previously characterized genes
with unknown roles in muscle development, and predicted genes of unknown
function, were found. These studies of newly identified genes revealed new patterns of gene
expression restricted to one of the two types of myoblasts, and also striking
muscle phenotypes. Whereas genes such as phyllopod play a crucial
role during specification of particular muscles, others such as
tartan are necessary for normal muscle morphogenesis (Artero, 2003).
The Toll10b mutation gives rise to embryos
composed primarily of somatic mesoderm. In these embryos FCs and FCMs are
readily detected, and they respond to the Ras and Notch signaling pathways
in the same way as their wild-type counterparts. Advantage was taken of this
fact to enrich Toll10b mutant embryos for FCs or FCMs,
which allowed a concentration on the transcription in these two specific
cell types within the context of the entire embryo. Genes known to be
expressed and regulated in FCs or FCMs emerged from the screen in the proper
categories. Not all known FC/FCM genes were detected in the screen for several
reasons: the high stringency set for interpretation of the array data; the
presence of only about one-third of the genome on the arrays; the loss of Dpp
in the Toll10b background, and the specific window of
myogenesis (5- to 9-hours) that was the focus of this investigation. However, a plethora
of potential new muscle regulators were uncovered, including known genes with
no previously recognized function in the mesoderm (such as phyl and
asteroid), and genes predicted from the Drosophila genome
sequence but not previously analyzed (Artero, 2003).
Various tests were applied to ascertain the validity of the results.
Available databases were analyzed to find evidence
that the known and predicted genes were expressed at the correct time and place. In
addition, Northern analysis with eleven genes tested the reliability of the
microarray detection and selection criteria; the results from all
genes tested agreed with the array data (Artero, 2003).
A Toll10b sample on the Northern blots allowed
ascertainment of why a gene is enriched in a particular condition. For example, in
the case of FC enriched genes, the signal in the Ras and
Notch lanes can be compared with Toll10b alone to
determine whether the Ras/Notch ratio for a gene is due to activation by Ras
or repression by Notch. Those genes that are 'enriched
under Notch conditions', for example, could reflect a variety of transcription
mechanisms that would result in a ratio of less than 0.6. By Northern
analysis, many of the 'Notch-regulated' genes, and hence the predicted
FCM genes, were found to be repressed by Ras signaling and slightly activated by Notch. As
a case in point, hibris is induced by Notch (2-fold)
and repressed by Ras (10-fold), both by Northern analysis and by in situ
hybridization in embryos (Artero, 2003).
A combination of in situ hybridization, immunostaining and confocal
microscopy was used to verify that the differential expression changes that were observed
in these overexpression embryos reflected true differential expression in the
wild-type situation. The expression of nine genes from different
functional categories was analyzed. For
seven of these, expression was detected in the predicted type of myoblast. For
two, asteroid (ast) and cadmus, no specific staining in embryos was detected by in situ hybridization. For
those genes that fell into the category of 'specific role in muscle
development uncertain', in situ hybridization of several (28%) showed
expression in tissues other than somatic mesoderm that are present in the
Toll10b background. These genes changed their expression
levels in response to Ras or Notch, and may be Ras and Notch targets in
non-mesodermal tissues (Artero, 2003).
The most stringent test, mutational analysis, was applied to a set of genes
for which mutations are available. Preliminary analyses of another four FCM-enriched genes was carried out: Elongation factor Tu mitochondrial (EfTuM), Glutamine synthetase 1, cadmus and parcas. All
four mutants have muscle defects, including muscle losses and aberrant muscle
morphologies. Thus all the genes tested show some muscle defect, supporting the usefulness of the genetic and genomic approach (Artero, 2003).
Taken together, these data suggest that the majority of genes detected play important roles in FCs or FCMs during muscle development. Some of these genes might not have been found in traditional forward genetic screens because of partial or
complete genetic redundancy. The data complement traditional forward genetic
approaches for finding genes crucial for muscle morphogenesis (Artero, 2003).
Each of the thirty FCs per abdominal hemisegment is hypothesized to produce
its own unique combination of transcriptional regulators, though the evidence
for this is limited. In turn the combination of regulators would control the
morphology of the final muscle. Although several transcriptional regulators
have been linked to FC identity, the molecular description is far from
complete. This screen contributed two more FC-specific genes. Previously known
markers, such as slouch or eve, once induced in the muscle
FC, are maintained throughout the remainder of development. Ubx,
which emerged from this screen, is a similarly simple case, as its transcripts
are steadily present in most FCs. By contrast, more
complex patterns of gene expression have been identified in FCs, such as the transient transcription of asense in a subset of FCs. The subsequent
transcriptional inactivation of asense may underlie temporal changes in
cell properties (Artero, 2003).
Even less is known about transcriptional regulators controlling FCM
differentiation. Only one gene, lame duck, has been shown to have a
role in FCMs. This screen has uncovered three more potential players: delilah,
E(spl)mß and CG4136, confirming that FCMs follow their own,
distinct, myogenic program. Discovering what aspects of FCM biology are
controlled by these transcriptional regulators awaits analysis of the
loss-of-function phenotypes (Artero, 2003).
Notch and Ras signaling pathways interact during muscle progenitor
segregation. The results suggest that phyl and
polychaetoid (pyd) may be additional links between the two
signaling pathways in FCs. phyl and pyd both interact
genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during
specification of SOPs and their progeny. This study
shows a similar regulation in muscle cells, where Notch signaling represses
phyl expression and Ras signaling increases phyl expression. Likewise, in the
nervous system, the segregation of SOPs requires pyd, a Ras target
gene, to negatively regulate ac-sc complex expression. Similarly, Pyd
may restrict the muscle progenitor fate to a single cell, perhaps by
regulating lethal of scute transcription. Thus, Pyd would collaborate
with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).
FCMs appear to integrate Ras and Notch signaling differently. Two genes
whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).
Embryos that lack or ectopically express phyl have morphological
defects in specific muscles, for example, in LL1 and DO4 in response to
diminished phyl function, and in DT1 and LT4 in response to increased
phyl function. The morphological defects in the loss-of-function
embryos appear to be due to a failure to specify particular FCs, a conclusion
that is based upon missing or abnormal production of the FC marker Kr. In eye
development and SOP specification, Phyl directs degradation of the
transcriptional repressor Tramtrack. In a subset
of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack
for degradation. The presence of Tramtrack would contribute to the specific
identity program of the muscle. Since Tramtrack is expressed in the mesoderm,
this possibility is likely. Alternatively, Phyl may be required for targeted
degradation of some other protein in a subset of FCs. The molecular partner
for Phyl during muscle differentiation is unknown, although preliminary data
suggest that sina is also expressed in somatic mesoderm and thus may
be its partner. These studies have
identified a new role for Phyl in muscle progenitor specification and suggest
the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).
A role for ubiquitination in muscle differentiation is further reinforced
by the identification of the RING finger-containing protein Goliath (Gol), induced by
activated Notch conditions, and CG17492, induced by activated Ras conditions.
Several RING-containing proteins function as E3 ubiquitin ligases, with the
ligase activity mapping to the RING motif itself. Ligase function has been experimentally confirmed for the Gol ortholog GREUL1
in Xenopus. Thus, targeted protein degradation during muscle morphogenesis could serve a host of crucial functions, such as protein turnover, vesicle sorting, transcription factor activation and signal degradation (Artero, 2003).
The simplest view of the 'founder cell' hypothesis is that each FC contains
all the information for the development of a particular muscle. By contrast,
FCMs have been seen as a naïve group of myoblasts, entrained to a
particular muscle program upon fusion to the FC. This work indicates that these
two groups of myoblasts have distinct transcriptional profiles. These data
raise the possibility of a greater role for FCMs in determining the final
morphology of the muscle and emphasize a need to characterize fully those FCM
genes. For
example, this screen identified a protein kinase of the SR splice site selector
factors (SRPK) whose transcripts are enriched in FCMs, suggesting that
regulation of the splicing machinery is important for muscle morphogenesis.
The Mhc gene undergoes spatially and temporally regulated alternative
splicing in body wall muscles conferring different physiological properties on
these muscles. This FCM-specific expression of SRPK may indicate that the
production of a particular Mhc isoform is regulated by the FCMs that
contribute to that muscle, rather than by the particular FC that seeds the
muscle. In addition, a number of observations suggest that FCMs may be a
diverse population of myoblasts, with different subsets having different
potential to contribute to the final muscle pattern. For example, hbs
expression suggests that only a subset of FCMs express the gene, and
twist expression in lame duck mutant embryos persists in a
subset of FCMs. This study provides additional genes
for exploring whether FCMs are a heterogeneous population of myoblasts as well
as determining the nature of FCM contribution to the final muscle (Artero, 2003).
The molecular events underlying complex morphological changes, such as
migration, cell fusion, cell shape changes or changes in the physiology of a
cell, require a rich and dynamic program of transcription changes. This study has described approximately one-third of this transcriptional profile. The FC- or
FCM-specific transcription of seven genes, and the mutant phenotype of four
selected genes, allowed the definition of new muscle mutations that
specifically affect the morphological traits of a subset of muscles (Artero, 2003).
The mechanisms underlying the setting of myotubes and choice of myotube number in adult Drosophila were examined. The pattern of adult myotubes is prefigured by a pattern of dumbfounded (duf) as determined by examining duf-lacZ-expressing myoblasts at appropriate locations. Selective expression of duf-lacZ in single myoblasts emerges from generalized, low-level expression in all adult myoblasts during the third larval instar. The number of founders, thus chosen, corresponds to the number of fibers in a muscle. In contrast to the embryo, the selection of individual adult founder cells during myogenesis does not depend on Notch-mediated lateral inhibition. These results suggest a general mechanism by which multi-fiber muscles can be patterned (Dutta, 2004).
In the Drosophila embryo, the diversification of muscle forming
mesoderm into founders and fusion-competent cells occurs through a process of
lateral inhibition mediated by Notch. Since single duf-lacZ-expressing cells are selected and appear to act as founder myoblasts during adult myogenesis, it is important to show whether, as in the embryo, a Notch-dependent lateral inhibition pathway mediates this selection process. To test whether Notch has a function in selecting specific myoblasts for duf-lacZ expression, a dominant-negative and a constitutively active form of Notch was used. It was reasoned that if lateral inhibition were involved, then overexpression of a dominant-negative form of Notch (dnNotch) in adult myoblasts would lead to an increase in the number of duf-lacZ-expressing founders, whereas overexpression of the active form (Nintra) should suppress duf-lacZ expression altogether (Dutta, 2004).
In fact, the results of these experiments appear to be contradictory:
thus, expression of UAS-dnNotch caused no change in the number of DVM
founders and flies of
the genotype 1151GAL4;UAS-dnNotch had the correct number of DLM and
DVM fibers. This conclusion was verified by reducing Notch
function in two additional ways. Function in the Notch signalling
pathway in myoblasts was reduced by overexpressing truncated forms of the protein
Mastermind (Mam), an essential component of the Notch signalling pathway. Mam
interacts with the intracellular domain of Notch and with Suppressor of
Hairless, and forms a transcriptional activation complex. Two
truncated versions of Mam, MamH and MamN, when overexpressed by the GAL4-UAS
system behave as dominant-negative proteins and elicit Notch loss-of-function
phenotypes. Overexpression of either UASMamN or UAS-MamH
in myoblasts using 1151-GAL4 had no effect on the
number of DVM founders. The role of Notch was further examined by
using a conditional allele, Nts1. Because of the close proximity of the duf and Notch loci, duf-lacZ, Nts
recombinants could not be generated and hence 22C10 was used as the marker for founder cells in the abdomen. The earliest time at which myoblasts expressing high levels of duf-lacZ are also labeled with 22C10 is at 24 hours APF. Notch
function was removed for different periods (2 hours, 4 hours, 6 hours and 8 hours) before this stage by raising Nts animals to the non-permissive
temperature, and the number of 22C10-stained cells associated with
the abdominal nerves was determined. The numbers of 22C10-expressing cells in the dorsal or lateral segments of the abdomen were determined and shown to be unaffected in
these experiments. It is known that all three approaches -- whether using the
dominant-negative Notch or mastermind constructs, or using
Nts animals -- are effective, since they all can reduce the
levels of Twist expression in adult myoblasts, a known
consequence of Notch reduction in adult myoblasts (Dutta, 2004).
Taken together, these results suggest that Notch is not required for the
selection of duf-lacZ-expressing myoblasts. However, in the converse
experiment, expression of Nintra in the myoblasts does suppress the formation
of founders, as would be expected for a selection mechanism based on lateral
inhibition. How can
these apparently contradictory findings be reconciled? Earlier studies have shownthat Notch acts as a positive regulator of Twist in the
myoblast population. Thus, Nintra expression in adult myoblasts leads to
maintained expression of Twist in these cells and to a failure of muscle
differentiation. If the absence of founders that was observe is a consequence
of this sustained expression of Twist in the myoblasts, then it would be expected
that simply expressing Twist constitutively in the myoblasts would mimic the
activated-Notch phenotype. Using 1151-GAL4 to drive Twist expression
in the adult myoblasts, it was found that at 12 hours APF there were no DVM
founders, suggesting that a decline in Twist expression, which is antagonized by the action of Nintra, is a requirement for elevated duf-lacZ expression. Indeed, founders are the first cells in the myoblast pool to show declining levels of Twist expression, with the result that the duf-lacZ-expressing
founders and Twist-expressing myoblasts are mutually exclusive cell
populations (Dutta, 2004).
During the formation of the Drosophila heart, a combinatorial network that integrates signaling pathways and tissue-specific transcription factors specifies cardiac progenitors, which then undergo symmetric or asymmetric cell divisions to generate the final population of diversified cardiac cell types. Much has been learned concerning the combinatorial genetic network that initiates cardiogenesis, whereas little is known about how exactly these cardiac progenitors divide and generate the diverse population of cardiac cells. In this study, the cell lineages and cell fate determination in the heart have been examined by using various cell cycle modifications. By arresting the cardiac progenitor cell divisions at different developing stages, the exact cell lineages for most cardiac cell types have been determined. Once cardiac progenitors are specified, they can differentiate without further divisions. Interestingly, the progenitors of asymmetric cell lineages adopt a myocardial cell fate as opposed to a pericardial fate when they are unable to divide. These progenitors adopt a pericardial cell fate, however, when cell division is blocked in numb mutants or in embryos with constitutive Notch activity. These results suggest that a numb/Notch-dependent cell fate decision can take place even in undivided progenitors of asymmetric cell divisions. By contrast, in symmetric lineages, which give rise to a single type of myocardial-only or pericardial-only progeny, repression or constitutive activation of the Notch pathway has no apparent effect on progenitor or progeny fate. Thus, inhibition of Notch activity is crucial for specifying a myogenic cell fate only in asymmetric lineages. In addition, evidence is provided that whether or not Suppressor-of-Hairless can become a transcriptional activator is the key switch for the Numb/Notch activity in determining a myocardial versus pericardial cell fate (Han, 2003).
Previous studies have suggested that Notch activity controls two distinct processes during the specification of cardiac cell fates. 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. Subsequent to the progenitor specification, Notch is required again for the specification of alternative cell fates of sibling cells produced during asymmetric cell divisions. In this
study, the cell autonomy of Notch was examined, by using eme-Gal4 (which confers expression in the mesodermal Eve lineage) to drive
activated forms of Notch and Su(H) exclusively in the mesodermal Eve lineages. Conditional ubiquitous expression of activated Notch was used to examine its lineage-specific function in other cardiac lineages. Notch was found to be 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 does 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) (Han, 2003).
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 an 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 one would expect to observe the same lineage phenotype as of Notch alone, i.e., a myogenic cell fate, since the primary role of Numb is to inhibit Notch signaling. Unfortunately, analysis of such double mutants is complicated by the earlier role of Notch in lateral inhibition (Han, 2003).
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, since production of the ligand is usually inhibited within the cell that experiences Notch signaling. In the asymmetric MP2 lineage of the Drosophila CNS, for example, ligand production appears to be required
in cells outside the MP2 lineage. A similar scenario may be operating in the asymmetric cardiac lineages (Han, 2003).
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. In
support of this model, it has been found 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, that prevent Su(H)-dependent transcriptional activation in
the absence of Notch signaling (Han, 2003).
The role of the PTB-containing, membrane-associated protein Numb in
preventing Notch activation in the nervous system is well established. To explore at which level Notch signaling is inhibited by
Numb in the cardiac lineages, numb was overexpressed 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 Notch 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. 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. Experiments described here 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 (Han, 2003).
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. As in the Eve and Svp lineage of the Drosophila heart, activation of the Notch pathway decreases 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 this study]. 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 (Han, 2003 and references therein).
Drosophila larval hemocytes originate from hematopoietic organs called lymph glands. These are composed of paired lobes located along the dorsal vessel. Two mature blood cell populations are found in the circulating hemolymph: the macrophage-like plasmatocytes, and the crystal cells that contain enzymes of the immune-related melanization process. A third class of cells, called lamellocytes, are normally absent in larvae but differentiate after infection by parasites too large to be phagocytosed. Evidence is presented that the Notch signaling pathway plays an instructive role in the differentiation of crystal cells. Loss-of-function mutations in Notch result in severely decreased crystal cell numbers, whereas overexpression of Notch provokes the differentiation of high numbers of these cells. In this process, Serrate, not Delta, is the Notch ligand. In addition, Notch function is necessary for lamellocyte proliferation upon parasitization, although Notch overexpression does not result in lamellocyte production. Finally, Notch does not appear to play a role in the differentiation of the plasmatocyte lineage. This study underlines the existence of parallels in the genetic control of hematopoiesis in Drosophila and in mammals (Duvic, 2002).
Drosophila hematopoiesis occurs in two distinct phases; one occurs during embryonic development and a second occurs in larvae. In embryos, a population of blood cells (hemocytes) differentiates in the head mesoderm then migrates to colonize the whole organism. These cells exhibit macrophagic activity and are called plasmatocytes. A second population of hemocytes, the crystal cells, differentiate simultaneously in the region of the anterior midgut. In larvae, hematopoiesis takes place essentially in so-called lymph glands, which are composed of a variable number (2-6) of paired lobes distributed along the dorsal vessel. The circulating hemolymph of larvae contain three fully differentiated hemocyte types: plasmatocytes, the professional phagocytes, represent the majority of the cells; crystal cells, which contain the enzymes necessary for immune-related melanization reactions and represent less than 5% of the cells; and lamellocytes, which are essentially devoted to encapsulation of large-sized invading parasites and are produced upon parasitization. At metamorphosis, the lymph glands degenerate, and, in adults, only the plasmatocyte population persists (Duvic, 2002).
A genetic dissection of hematopoiesis has shown that hemocyte differentiation in embryos is dependent on several transcription factors: blood cell fate is determined by the GATA factor Serpent (encoded by srp), plasmatocyte identity is specified by the zinc-finger transactivator Glial Cells Missing (gcm), and crystal cell identity is specified by the Runt domain AML1-related Lozenge transactivator (lz). The Friend-of-Gata homolog, U-shaped (ush), antagonizes crystal cell development. In larvae, a similar approach has shown that gcm and lz, respectively, control plasmatocyte and crystal cell differentiation. srp is expressed early in larval lymph gland cells, and it is assumed, although not proven, that it has the same function as in embryonic hematopoiesis (Duvic, 2002).
The potential role of N in Drosophila hematopoiesis was addressed by taking advantage of the temperature-sensitive Nts1 allele. At a permissive temperature, Nts1 larvae have a wild-type number of circulating plasmatocytes and crystal cells. However, when second instar larvae were shifted to the restrictive temperature (29°C), it was observed that (at the wandering stage) the mutant larvae exhibited a strong reduction (up to 60%) in the number of crystal cells. Plasmatocyte numbers are not affected. This analysis was shifted by using a transgenic line in which a cDNA sequence encoding a ligand-independent constitutively active form of N (Nic) was placed under the control of UAS elements. Overexpression of Nic with an ubiquitous Gal4 driver (hsp-Gal4) results in a dramatic increase in the number of crystal cells in larvae (up to 7-fold). Similar high numbers of crystal cells were recorded in larvae carrying the gain-of-function allele of Notch NMcd8. Altogether, these results indicate that the function of N is mandatory for crystal cell differentiation in larval development. Since plasmatocyte numbers remain wild-type in these experiments, the observed phenotypes do not reflect a generalized effect on blood cell proliferation, but they are specific to one lineage, i.e., the crystal cells (Duvic, 2002).
Two well-established downstream effectors of the Notch signaling pathway, Suppressor of Hairless [Su(H)] and Deltex (Dx), were examined for their involvement in hematopoiesis. A larval viable Su(H) interallelic combination was generated and mutant third instar larvae were observed to be almost totally devoid of crystal cells; however, they had wild-type plasmatocyte counts. In contrast, Dx (in Dx1 and DxP strong hypomorphic alleles) mutant blood cell counts were normal for both cell types, suggesting that the effect of N on crystal cell production is Dx independent (Duvic, 2002).
Crystal cells differentiate within the larval hematopoietic organ, the lymph glands, and the effect of the various mutations on differentiation in situ were examined. For this, an antibody raised against dipteran prophenoloxidase (proPO), a zymogen required for melanization reactions, was examined. In Drosophila, proPO is produced and stored in crystal cells and is released during host defense reactions. It was first noted that, in wild-type larvae, proPO is synthesized during the differentiation of crystal cells within the hematopoietic organ. Interestingly, a gradient of differentiation is apparent within the successive lymph gland lobes along the anteroposterior axis: the anteriormost lobes contained numerous proPO-positive cells, whereas the adjacent, more posterior lobes contain no or few positive cells and the most posterior lobes are totally devoid of differentiating crystal cells. In Nts1 mutants placed at a restrictive temperature, the number of proPO-positive cells is significantly reduced in the lymph glands, and they were occasionally totally absent. A similar phenotype was observed in a Su(H) and in a Ser mutant context. Conversely, activation of the Notch pathway in NMcd8 larvae and in larvae carrying UAS-Nic, UAS-Ser, or UAS-Dl transgenes driven by hsp-Gal4 dramatically increased the number of proPO-positive cells in lymph glands. Not only are the anteriormost lobes packed with such cells, but more posterior lobes are often found to contain large numbers of differentiating crystal cells. As expected, similar phenotypes are obtained when the e33C-Gal4 driver is used; this driver is strongly expressed in the lymph glands (Duvic, 2002).
The GATA factor Srp determines the hemocyte fate in Drosophila.
Since srp is expressed in the larval lymph glands, it was asked whether this expression was affected in an N mutant context. Nts1 larvae raised at the restrictive temperature were examined. The expression of srp is wild-type, indicating that the effect of Notch signaling on crystal cell differentiation occurs downstream of srp function (Duvic, 2002).
In Drosophila larvae, an additional blood cell type, the lamellocyte, is normally not present in healthy individuals. Lamellocyte differentiation is triggered by immune conditions such as infestation by parasitic wasps that lay eggs in second instar larvae. Lamellocytes, together with crystal cells, participate in the encapsulation of wasp eggs, which are eventually killed within the melanized capsules. Lamellocyte production is initiated a few hours after wasp egg laying, and 48 hr later, lamellocytes represent an important proportion of circulating hemocytes. Cellular reactions to wasp parasitization were examined in Nts1 larvae placed at the restrictive temperature. Although the production of lamellocytes was not totally abolished in the mutant larvae, it was significantly reduced compared to wild-type larvae or to Nts1 larvae that had been maintained at the permissive temperature. Thus, the absence of N function prevents normal lamellocyte differentiation in response to wasp parasitization. However, overexpression of Nic does not result in lamellocyte production, in contrast to crystal cells (Duvic, 2002).
These data indicate that the Notch signaling pathway plays an instructive role in the specification of the crystal cell lineage in Drosophila larvae. This role is distinct from that reported for the lz gene. Indeed, although lz function is mandatory for crystal cell differentiation, its overexpression per se has no phenotype with regard to crystal cells. Notch signaling has been extensively analyzed in the context of mammalian hematopoiesis. In particular, Notch1 prevents the differentiation of pluripotent stem cells through stimulating expression of the GATA-2 transactivator. This picture somewhat contrasts with that observed in Drosophila, in which N is clearly not involved in maintaining pools of undifferentiated blood cell precursors and does not regulate the expression of the GATA factor srp. At later stages of mammalian hematopoiesis, Notch1 controls lymphoid cell fate specification, namely, by favoring T cell commitment over that of B cells. This conceptually parallels the role of N in Drosophila hematopoiesis, in which it favors crystal cell commitment and differentiation (Duvic, 2002).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Notch:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions | Post-transcriptional regulation of Notch mRNA
| Developmental Biology
| References
Society for Developmental Biology's Web server.