pebbled
pebbled expression in the midgut is controlled by the maternal and zygotic members of the torso mediated terminal pathway. Embryos produced by homozygous torso loss-of-function mutant females lack Hnt protein in the posterior midgut, which lies within the domain of torso function. Instead of extending their germ bands dorsoanteriorly, most such embryos form spiralled germ bands. Reciprocally, embryos carrying torso gain of function mutations lack dorsal expression (that is, in the presumptive amnioserosa), consistent with conversion of central cell fates to more terminal ones. These embryos also show expanded expression of Hnt protein in the enlarged posterior midgut primordium and a twisted gastrulation phenotype (Yip, 1997).
tailless mutations have little effect on pebbled expression; from analysis of huckebein tailless double mutants, it is clear that the only loss of Hnt protein expression in tailless mutants occurs in the region from which the Malpighian tubule primordia originate, consistent with the reported role for tll and peb in the development of these structures. hkb mutant embryos lack Hnt protein expression in the regions from which the anterior and posterior midgut normally arise; expression remains only in the presumptive ureter of the Malpighian tubules. In hkb tll double mutant embryos, Hnt protein is not present at all in the domains that would form anterior and posterior midgut and Malpighian tubule primordia; however expression does occur in the amnioserosa. Germ-band retraction occurs in tll or hkb single mutants as well as in hkb tll double mutants, suggesting that midgut expression of Hnt is not necessary for germ-band retraction (Yip, 1997).
Hnt protein is present in u-shaped, tailup and Egfr mutants. These results suggest that peb either resides upstream of these three genes in the same hierarchy or one or more of these genes functions in a parallel pathway. In contrast, endodermal expression of Hnt is missing in serpent mutant embryos. This last result is consistent with the fact that serpent is required to establish the identity of the endodermal midgut; loss-of-function mutations in serpent result in transformation of endoderm into ectoderm (Yip, 1997 and references).
pebbled expression in the amnioserosa is regulated by the dorsoventral pathway. Dorsal Hnt protein expression is reduced in genetically ventralized mutant embryos such as those produced by saxophone or cactus null females. Reciprocally, dorsal Hnt expression expands ventrally in dorsalized embryos. Anterior midgut expression of Hnt is also affected by the dorsoventral pathway (Yip, 1997).
The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial
tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal
development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act
as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal
specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh
and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression
requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some
downstream genes but not others. While tracheal expression of tkv depends on
vvl, it appears to be independent of trh. The opposite
appears to be the case for two other tracheal genes, tracheal defective (tdf) and pebbled (peb) [also known as hindsight
(hnt)], which code for two putative transcription factors. Both genes
appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes
seem to be common targets of vvl and trh but others seem
to depend only on one of them (Boube, 2000).
The dorsal ectoderm of the Drosophila embryo is
subdivided into different cell types by an activity gradient
of two TGFbeta signaling molecules, Decapentaplegic
and Screw. Patterning responses to this gradient
depend on a secreted inhibitor, Short gastrulation
and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a
number of Dpp target genes has been examined in transgenic embryos that
contain ectopic stripes of Dpp, Sog and Brk expression.
These studies suggest that the Dpp/Scw activity gradient
directly specifies at least three distinct thresholds of gene
expression in the dorsal ectoderm of gastrulating embryos.
Brk was found to repress two target genes, tailup/islet and
pannier, that exhibit different limits of expression within
the dorsal ectoderm. These results suggest that the Sog
inhibitor and Brk repressor work in concert to establish
sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target
genes depends on the Drosophila homolog of the CBP
histone acetyltransferase (Ashe, 2000).
Different dorsal ectoderm genes were examined in a variety of
mutant and transgenic embryos using digoxigenin-labeled
RNA probes and in situ hybridization. The
normal expression patterns suggest the occurrence of at least
three thresholds of gene activity in response to the Dpp/Scw
activity gradient. The Race and hindsight/pebbled (hnt) genes are expressed
in narrow strips in the dorsalmost regions of the embryo, although the anteroposterior limits of the
two patterns are distinct. It is conceivable that early-acting
segmentation genes are responsible for repressing Race in
posterior regions and hnt in anterior regions. Somewhat
broader expression patterns are observed for tup and ush. These patterns encompass the presumptive amnioserosa
and dorsal regions of the dorsal epidermis.
Broad staining patterns are observed for two genes encoding
GATA transcription factors, dGATAc and pnr. pnr is expressed throughout the dorsal
ectoderm in the presumptive thorax and abdomen.
dGATAc exhibits a nearly reciprocal pattern in anterior and
posterior regions; staining is mainly excluded from regions
expressing pnr, although a weak patch of staining is detected
in a portion of the presumptive amnioserosa. Most of the
subsequent analyses on gradient thresholds have focussed on the
regulation of hnt, tup and pnr (Ashe, 2000).
All of the aforementioned genes are virtually silent in the
dorsal ectoderm of dpp-/dpp- embryos, while
changes in dpp+ gene dose cause altered patterns of expression. For example, increasing the number of dpp+ copies
from two to three to four results
in a sequential expansion of the hnt expression pattern, whereas
expression is lost in dpp/+ heterozygotes. In
contrast, ush is expressed in dpp/+ heterozygotes, although
there is a marked narrowing in the expression pattern as
compared with wild-type embryos.
Three copies of dpp+ cause an expansion of the ush pattern. Similarly, the tup expression pattern is narrower in
dpp/+ heterozygotes and expanded in embryos with three
copies of dpp.
Further evidence that hnt and ush are early targets of the Dpp
signaling pathway was obtained by analyzing transgenic
embryos that contain the dpp-coding sequence attached to the
eve stripe 2 enhancer. These embryos exhibit both
the endogenous dpp pattern in the dorsal ectoderm as well as an ectopic stripe of expression (Ashe, 2000).
Additional Dpp/Scw target genes were examined for
repression by the stripe2-brk transgene. Those showing altered
patterns of expression include tup, rho, hnt and Race. The normal tup expression pattern
encompasses both the presumptive amnioserosa and dorsal
regions of the dorsal epidermis. In transgenic
embryos, there is a gap in the pattern in regions where the
stripe2-brk fusion gene is expressed. These results
suggest that Brk represses tup, even though it appears to
respond to a different threshold of Dpp/Scw signaling than pnr.
Additional experiments were done to determine whether Brk
directly represses tup expression, or works indirectly by
inhibiting Dpp signaling (Ashe, 2000).
The limits of the tup expression pattern seem to depend on
both Sog and Brk. The introduction of the stripe2-brk transgene leads to a clear gap in the tup expression pattern, although there is a narrow stripe of repression in gd- mutants lacking the transgene. The stripe2-sog
transgene causes a more extensive gap in the tup pattern. The stripe2-brk transgene was also found to repress Race,
hnt and rho in this assay (Ashe, 2000).
A summary is presented of Dpp signaling thresholds in the embryo. The Dpp/Scw activity
gradient presumably leads to a broad nuclear gradient of Mad and
Medea across the dorsal ectoderm of early embryos. It is conceivable
that the early lateral stripes of brk expression lead to the formation of
an opposing Brk repressor gradient through the limited diffusion of
the protein in the precellular embryo. Peak
levels of Dpp and Scw activity lead to the activation of Race and hnt
at the dorsal midline. The tup and ush patterns represent another
threshold of gene activity. The similar patterns might involve
different mechanisms of Dpp signaling since tup is repressed by Brk,
whereas ush is not. Finally, the broad pnr pattern
represents another threshold of gene activity. It is not inhibited by
Sog but is repressed by Brk. It is possible that tup and pnr are
differentially repressed by a Brk gradient. Low levels of Brk might
be sufficient to direct the lateral limits of the tup pattern, whereas
high levels may be required to repress pnr (Ashe, 2000).
Su(H)/CBF1 is a key component of the evolutionary conserved Notch
signalling pathway. It is a transcription factor that acts as a repressor in
the absence of the Notch signal. If Notch signalling is activated, it
associates with the released intracellular domain of the Notch
receptor and acts as an activator of transcription. During the development of
the mechanosensory bristles of Drosophila, a selection process called
lateral inhibition assures that only a few cells are selected out of a group
to become sensory organ precursors (SOP). During this process, the SOP cell is
thought to suppress the same fate in its surrounding neighbours via the
activation of the Notch/Su(H) pathway in these cells. Although
Su(H) is required to prevent the SOP fate during lateral inhibition, it is
also required to promote the further development of the SOP once it is
selected. Importantly, in this situation Su(H) appears to act independently of
the Notch signalling pathway. Loss of Su(H)
function leads to an arrest of SOP development because of the loss of
sens expression in the SOP. These results suggest that Su(H) acts as a repressor that suppresses the activity of one or more negative regulator(s) of sens expression. This repressor activity is encoded by one or several genes of the E(spl)-complex. These results further suggest that the position of the SOP in a proneural cluster is determined by very precise positional cues, which render the SOP insensitive to Dl (Koelzer, 2003).
Thus Su(H) is required to promote
SOP development. This is based on the fact that most cells of proneural
clusters in the notum that lack Su(H) function do not express SOP
markers such as Sens, Hindsight (Hnt) and partially neurA101-lacZ.
Loss of neurA101-lacZ expression has been attributed to a 'general sickness' of
the mutant discs, since the lack of neurA101-lacZ expression has only been observed in the late developing proneural clusters. The data argue against such an explanation: Presenilin (Psn)
mutant wing imaginal discs exhibit a stronger neurogenic phenotype than do
Su(H) mutants. Similar to Su(H) mutants, homozygous
Psn mutant animals also die during the early pupal phase.
Nevertheless, the cells of the proneural clusters of these mutants express all
tested markers, indicating that SOP development is not affected. The same is
true for kuzbanian (kuz) mutants, whose mutant phenotype is comparable with that
of Su(H) mutants. Hence, general sickness of the wing imaginal disc
cells is not likely to explain the arrest of SOP development in Su(H) mutants (Koelzer, 2003).
A role of Su(H) in development of the SOP is surprising, because it is a
core element of the Notch signalling pathway and the activity of this
pathway is required to prevent SOP development in cells of the proneural
clusters. Importantly, in this new role, Su(H) seems to function
independently of the Notch signalling pathway. This is indicated by the finding that the Su(H) mutant phenotype is epistatic over that of Psn mutants (Koelzer, 2003).
The data presented here indicate that Su(H) appears to be required to
suppress the activity of one or more members of the E(spl)-C, that in turn suppress the expression of genes such as hnt and sens. This conclusion is based on: (1) the failure of Su(H)VP16 to activate
sens; (2) the fact that Psn H double mutants display a
similar loss or reduction of sens expression as Su(H) and
Su(H); Psn double mutants, and (3) the fact that expression of a
Su(H) construct that is unable to bind H (UAS Su(H)DeltaH)
leads to an arrest of SOP development in Psn mutant wing imaginal
discs. Several reports show that H is involved in Su(H)-related
suppression of gene expression in the absence of Notch signalling. Recently, it has been shown that H acts as a bridge between
Su(H) and the general co-repressors CtBP and Gro. It is therefore likely, that this Su(H)/H/Gro/dCtBP complex mediates the repressor function required during SOP development (Koelzer, 2003).
Repression by Su(H) is not strictly required in all proneural clusters to
allow expression of sens and other late SOP markers. Examples are the
clusters in the wing region, such as the clusters of the dorsal radius.
However, even in these clusters, sens and hnt are not
expressed in all cells that express early markers, such as
neurA101. Therefore, it appears that the activity of Su(H) promotes SOP development also in these clusters. The clusters of the dorsal radius give
rise to other types of sense organs, such as companiforme sensilla, and it is
possible that there are different requirements for the activity of
Su(H) for the development of the different types of sense organs (Koelzer, 2003).
In Su(H) mutant cell clones induced during the first
larval instar stage, hnt is expressed in a fraction of cells of
specific proneural clusters, such as the scutellar cluster, but absent or
strongly reduced in other clusters. It was further found that in Su(H)
mutant wing imaginal discs, expression of sens and hnt is either
lost or strongly reduced when compared to mutant cell clones induced during the first larval instar (Koelzer, 2003).
Altogether, these observations suggest that the Notch pathway
might have two separable functions during SOP development. During early phases of a proneural cluster, the activity of the pathway keeps the cells of the cluster undecided, perhaps by mutual repression. Owing to positional cues, one cell becomes insensitive to the inhibitory signal and adopts the SOP fate. Subsequently the SOP inhibits its immediate neighbours by sending an inhibitory signal through Dl (Koelzer, 2003).
Absence of Krüppel at stage 11 correlates with the premature apoptosis of the differentiated amnioserosa in pebbled mutants (Frank, 1997).
pebbled may regulate Krüppel. Krüppel, which accumulates in wild-type embryos in the nuclei of amnioserosal cells, is absent from most but not all of these cells in stage 11 peb mutants (Yip, 1997).
As the germ band shortens in Drosophila melanogaster embryos, cell shape changes cause segments to narrow anteroposteriorly and to lengthen dorsoventrally. One
of the genes required for this retraction process is the hindsight (hnt) gene. hnt encodes a nuclear Zinc-finger protein that is expressed in the extraembryonic
amnioserosa and the endodermal midgut prior to and during germ band retraction. Through analysis of hnt genetic mosaic embryos, it has been shown that hnt activity in the amnioserosa, particularly in those cells that are adjacent to the
epidermis, is necessary for germ band retraction. In hnt mutant embryos the amnioserosa undergoes premature cell death. Prevention of premature apoptosis in hnt mutants does not rescue retraction. Thus, failure of this process is not
an indirect consequence of premature amnioserosal apoptosis; instead, hnt must function in a pathway that controls germ band retraction (Lamka, 1999).
The Kruppel
gene is activated by hnt in the amnioserosa while the Drosophila insulin receptor (INR) functions downstream of hnt in the germ band. Kr protein is first detected in the amnioserosa of wild-type embryos during germ band extension; however, in hnt mutants, Kr is clearly absent from most amnioserosal cells by stage 11. Loss of Kr in hnt mutants is not a result of premature apoptosis of the amnioserosa. Rather the Kr gene resides downstram of Hnt in the hnt genetic hierarchy in the amniserosa. A specific role for Kr in germ band retraction remains to be defined. Using a heat shock insulin receptor transgene to overexpress the wild-type insulin receptor, nearly complete rescue of germ band retraction is observed in hnt mutants. The extent of rescue depends on the strength of the hnt allele used. The fact that high levels of the Inr can rescue germ band retraction in hnt mutants is consistent with the possiblility that the Inr functions downstream of hnt in a germ band retraction pathway. Evidence against a
physical model in which the amnioserosa 'pushes' the germ band during retraction is presented. Rather, it is likely that the amnioserosa functions in production, activation, or
presentation of a diffusible signal required for retraction (Lamka, 1999).
Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle (M/E switch). This study shows that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of cubitus interruptus. These studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation (Sun, 2007).
How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. These data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Sun, 2007).
Studies have shown that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature-cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed. The current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle (Sun, 2007).
The finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots, suggesting that an additional cell cycle also takes place. Further testing of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may not be the sole mediator of the Notch effect; for example, Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later, suggesting that Hnt function is also required for chorion-gene amplification (Sun, 2007).
The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells. Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots. The nuclear sizes of fu mutant cells were similar to those of the wild-type at the same developmental stage, and no fragmentation of the chromosomes was observed. Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus, Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of Ci-155 in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis. Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells (Sun, 2007).
Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells, but it is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt. Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Sun, 2007).
An interesting observation from these studies is that ttk clones have a phenotype similar to that of hnt clones. As in Notch regulation of Hnt, ttk is possibly downstream of Notch, but the current analysis of Notch mutants in stage-1 to stage-10 egg chambers showed no obvious change in Ttk expression. It was also found that Hnt has no role in regulating ttk expression. The findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis. The phenotypic similarity between hnt and ttk mutants suggests that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye. Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear (Sun, 2007).
Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt-deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process. Second, nurse-cell endoreplication does not require Hnt; no obvious defect was detected in hnt germline clones. The specific role of Hnt in follicle-cell-cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation (Sun, 2007).
Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on the cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway. Hh and Notch therefore act sequentially and positively during the SMW, whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis, but it is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling; mutation of the negative regulator of the Hh pathway, ptc, in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germline cells. These ptc mutant cells show no accumulation of Ci-155, consistent with the finding that Notch signaling suppresses ci transcription through Hnt. The ptcS2 cells that were out of contact with germline cells remained in the mitotic cycle because they could not receive Dl signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state (Sun, 2007).
Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration. When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% of egg chambers showed defects in border-cell migration. Notch signaling, as well as ttk, has been reported to be required for border-cell migration. Hnt was found to be expressed in the border cells and depended on Notch signaling. The occasional hnt border-cell clones observed also showed defects in border-cell migration, so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells (Sun, 2007).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
pebbled:
Biological Overview
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.