cubitus interruptus
The spatial pattern of expression of ci during development is unusual in that,
unlike most other segmentation genes, it exhibits uniform expression throughout cellular blastoderm
and gastrulation stages and does not resolve into a periodic pattern until the end of the fast phase of
germ-band elongation [Image] when it is present in 15 broad segmentally repeating stripes along the
anterior-posterior axis of the embryo. Registration of the ci stripes of expression relative to the
stripes of other segment polarity genes shows that ci is expressed in the posterior three-quarters of
every parasegment. This registration does not correlate with the pattern defects observed in ci
mutants (Orenic, 1990).
Expression of ci is observed in the hindgut primordia and in the head region corresponding to the procephalic lobe. It accumulates in the ectoderm, nervous system and mesoderm during germ band elongation. There is very little expression by stage 15 (Orenic, 1990).
In Drosophila embryos, segment boundaries form at the posterior edge of each stripe of engrailed expression. An HRP-CD2 transgene has been used to follow by transmission electron microscopy the cell shape changes that accompany boundary formation. The first change is a loosening of cell contact at the apical side of cells on either side of the incipient boundary. Then, the engrailed-expressing cells flanking the boundary undergo apical constriction, move inwards and adopt a bottle morphology. Eventually, grooves regress, first on the ventral side, then laterally. Groove formation and regression are contemporaneous with germ band retraction and shortening, respectively, suggesting that these rearrangements could also contribute to groove morphology. The cellular changes accompanying groove formation require that Hedgehog signalling be activated, and, as a result, a target of Ci is expressed at the posterior of each boundary (obvious targets like stripe and rhomboid appear not to be involved). In addition, Engrailed must be expressed at the anterior side of each boundary, even if Hedgehog signalling is artificially maintained. Thus, there are distinct genetic requirements on either side of the boundary. In addition, Wingless signalling at the anterior of the domains of engrailed (and hedgehog) expression represses groove formation and thus ensures that segment boundaries form only at the posterior (Larsen, 2003).
Segmental boundary formation is initiated shortly after germ-band retraction has begun. They are recognizable as periodic indentations in the epidermis that separate cells expressing engrailed at the anterior from those expressing rhomboid at the posterior. To allow identification of cells in electron micrographs, a transgenic membrane marker was devised based on horseradish peroxidase (HRP), which catalyses the production of an electron-dense product from diaminobenzidine (DAB). HRP was fused to the transmembrane protein CD2 so that the marker would outline cells and thus reveal cell shapes. This inert fusion protein was expressed under the control of engrailed-Gal4, so that the membrane of engrailed-expressing cells appears dark under the electron microscope (Larsen, 2003).
Engrailed has both a cell autonomous and
a non-cell autonomous function in the establishment of the compartment
boundary in wing imaginal discs. Although the compartment boundary does not
trace its embryonic origin to segment boundaries, there is
a striking parallel between the two. For segmental grooves
to form, Hedgehog signaling is required in cells at the posterior of the
boundary, even if engrailed expression is artificially maintained at
the anterior side. Conversely, Hedgehog signaling is not sufficient as
exogenous expression of hedgehog in the absence of engrailed
does not lead to groove formation (Larsen, 2003).
It is the cells that line the anterior side of segment
boundaries (the most posterior engrailed-expressing cells) that
undergo the most distinctive behavior during groove formation. This behavior
requires Hedgehog signalling, and yet engrailed-expressing cells are
not responsive to this signal. Therefore, their morphological changes must be
in response to a signal originating from neighboring non-engrailed
expressing cells. This could be achieved through standard paracrine signaling
or by contact-dependent signal mediated by cell surface proteins. Whatever the mechanism, Hedgehog-responsive cells influence the behavior of adjoining
engrailed-expressing cells across the boundary, and crosstalk between
the two cells takes place. This is reminiscent of the situation found during eye morphogenesis; at rhombomere
boundaries cross communication between neighboring rhombomere cells are required for rhombomere formation (Larsen, 2003).
Because boundaries form in the complete absence of Ci
(in ci94), it is concluded that the activator form of Ci is
not required for segment boundary formation. However, no boundary forms in
ciCell mutant embryos, indicating that the presence of
Ci[75] (the repressor) prevents boundary formation. It is suggest therefore that boundary formation requires the expression of a gene (x) that is
repressed by Ci[75] but does not require Ci[155] to be activated. Presumably,
an activator of x is constitutively present but, in the absence of
Hedgehog, it is prevented from activating x expression by Ci[75].
Hedgehog signaling would remove Ci[75] and thus allow activation to occur. Two characterized target genes of Hedgehog (wingless and
rhomboid) follow the same mode of regulation. For example, expression
of wingless in the embryonic epidermis decays in
ciCell but is still present in the complete absence of Ci,
in ci94 embryos (Larsen, 2003).
Although Hedgehog signaling is activated both at the anterior and the
posterior of its source, segment boundaries only form at the posterior. One
reason for this asymmetry is that Wingless signaling represses boundary
formation at the anterior. Indeed, in the absence of Wingless, boundaries are
duplicated, as long as expression of Engrailed and Hedgehog is artificially
maintained. It is concluded that expression of x is repressed by Wingless signalling. Two obvious candidates for x are Rhomboid and Stripe. Genes encoding both these proteins are activated by Hedgehog signaling and repressed by Wingless
signaling and, indeed, both are expressed in cells that line the
segment boundary. To determine if either gene could mediate the role of
Hedgehog in boundary formation, the respective mutants were examined. No effect
on grooves could be seen. It is concluded that neither rhomboid nor stripe is
required for boundary formation although the possibility
that these genes could contribute in a redundant fashion cannot be excluded. Overall the genetic
analysis suggests that additional targets of Hedgehog must be involved in
boundary formation. It will be interesting to find out whether any of these
targets will turn out to be implicated in compartment boundary maintenance as
well (Larsen, 2003).
Although the role of a Hedgehog target gene in boundary
formation has been emphasized, it is clear from this analysis that engrailed also has a
cell-autonomous role. Even though Engrailed
represses ci expression, its role in boundary formation is likely to
involve the transcriptional regulation of another target gene. One possibility is
that Engrailed could be a repressor of x and that boundaries would
form at the interface between x-expressing and non-expressing cells.
However, it is thought that instead, or in addition, Engrailed has a
Hedgehog-independent effect on cell affinity and that this could contribute to
boundary formation. Of note is the observation that
engrailed-expressing cells remain together in small groups even when
boundaries are lost for lack of hedgehog. This suggests that
engrailed-expressing cells have increased affinity for one another.
Thus, Engrailed could specify P specific cell adhesion independently of
Hedgehog. Clearly, future progress will require the identification of
Engrailed target genes that control such preferential affinity and/or
contribute to boundary formation (Larsen, 2003).
ci is expressed in the anterior compartment of the eye, antennal, leg, wing and haltere imaginal discs. In eye discs CI staining is elevated just anterior to the morphogenetic furrow. ci is also expressed in the larval brain lobes and along the midline of the ventral ganglia (Motzny, 1995).
Unlike the thoracic discs, the anterior and posterior compartmental organization of the genital
imaginal disc is compound, consisting of
three primordia the female genital, male genital, and anal primordia. Each primordium is divided into anterior and posterior compartments. Genes
that are known to be expressed in a compartment-specific manner in other discs (engrailed,
hedgehog, patched, decapentaplegic, wingless and cubitus interruptus) are expressed in
analogous patterns in each primordium of the genital disc. Specifically, engrailed and cubitus
interruptus are expressed in complementary domains, while patched, decapentaplegic and
wingless are expressed along the border between the two domains. en and inv are required in the posterior comparment of the genital disc to repress dpp and activate hh. Mitotic clones induced at
the beginning of the second larval instar do not cross the boundary between the
engrailed-expressing and cubitus interruptus-expressing domains, indicating that these
domains are true genetic compartments (Chen, 1997).
The adult clonal phenotypes of protein kinase A and engrailed-invected
mutants provide a more detailed map of the adult genitalia and analia with respect to the
anterior/posterior compartmental subdivision. A new model has been proposed to
describe the anterior and posterior compartmental organization of the genital disc. Each of the three primordia (female, male and anal) is composed of its own anterior and posterior compartments. Each primordium has a larger anterior compartment and a smaller posterior compartment. Each genital disc is divided into anterior and posterior compartment (Chen, 1997).
Very little information is available about gene expression during the larval period, a developmental interval critical to the formation of the adult. To what extent does gene expression during this period resemble that in the embryonic stages, and how does gene expression during the larval period contribute to segment polarity in the adult? In fact, all the genes expressed during embryonic segment polarity also play a similar role in the formation of the adult. Cells destined to form the cuticle of the adult abdomen are present as clusters of small, non-dividing diploid cells (the anterior dorsal, posterior dorsal and ventral histoblast nests) located at stereotyped postions in the larval epidermis. These cells, just as do their embryonic counterparts, express engrailed, hedgehog, wingless, patched, cubitus interruptus and sloppy paired in a stereotyped manner dependent on their positions within each segment. Each
segment is subdivided into an anterior (A) and posterior (P) compartment, distinguished by activity of the selector gene engrailed (en)
in P but not A compartment cells. The ventral epidermis of each abdominal segment forms a flexible cuticle, the pleura, with a small plate of sclerotised cuticle, the sternite, centered on the ventral midline. The pleura is covered with a uniform lawn of hairs, all pointed posteriorly, whereas the sternite contains a stereotyped pattern of bristles. Posterior compartments are to a large degree devoid of hairs and bristles, while the sternite cuticle of the A compartment consists of an anterior-to posterior progression of six types of cuticle distinguished by ornamentation and pigmentation. Just anterior to the posterior compartment, A6 is unpigmented, with hairs and none
of the larger ornaments called bristles. A5 is darkly pigmented with hairs and bristles of large size. A4 and A3 are darkly and lightly pigmented respectively with moderately sized hairs and bristles. A2 is lightly pigmented with hairs, and A1, adjacent to the next more anteriorly located "posterior" compartment is unpigmented without hairs (Struhl, 1997a).
Hedgehog (Hh), a protein secreted by engrailed expressing P compartment cells, spreads
into each A compartment across the anterior and the posterior boundaries to form opposing concentration gradients that organize cell
pattern and polarity. Anteriorly and posteriorly situated cells within the A compartment respond in distinct ways to Hh:
they express different combinations of genes and form different cell types. patched is expressed at both boundaries. patched is expressed in a graded fashion within each stripe, just anterior to each P compartment. ci peaks at high level in those cells abutting Hh- secreting cells of the P compartment and declines progressively in cells further away. wingless is also expressed in this domain and sloppy paired is expressed in the same region as wingless. decapentaplegic is expressed only in the ventral pleura in those A compartment cells neighboring P compartment cells within the same segment. dpp is not expressed immediately behind posterior compartments (Struhl, 1997).
sightless (sit) is required for the activity of Drosophila Hh in the eye and wing imaginal discs and in embryonic segmentation. sit acts in the cells that produce Hh, but does not affect hh transcription, Hh cleavage, or the accumulation of Hh protein. sit encodes a conserved transmembrane protein with homology to a family of membrane-bound acyltransferases. The Sit protein could act by acylating Hh or by promoting other modifications or trafficking events necessary for its function (Lee, 2001b).
One of the critical signals triggering photoreceptor development is Hedgehog (Hh), which is expressed at the posterior margin of the disc prior to differentiation and subsequently in the differentiating photoreceptors. Hh activates the expression of decapentaplegic (dpp) in a stripe at the front of differentiation, or morphogenetic furrow; Dpp signaling also promotes photoreceptor formation. dpp expression is lost from the morphogenetic furrow in sit mutant eye discs. Another target of Hh signaling, the proneural gene atonal, also requires sit for its expression. Despite this lack of Hh target gene expression, a hh-lacZ enhancer trap is expressed at the posterior margin of sit mutant eye discs, indicating that hh expression is established normally. This suggests that the sit phenotype could be due to a defect in Hh signaling (Lee, 2001).
Hh signaling has been extensively studied in the wing disc, where hh is expressed in the posterior compartment and signals to cells just anterior to the compartment boundary to upregulate the expression of dpp and patched (ptc). The Hh signal is mediated by the stabilization and activation of the full-length form of the transcription factor Cubitus interruptus (Ci). This stabilization can be detected with an antibody directed against the C-terminal region of Ci, which fails to recognize the cleaved form of Ci produced in the absence of Hh signaling. sit mutant wing discs show defects consistent with a lack of Hh pathway function; ptc expression is not upregulated at the compartment boundary, and dpp expression is almost completely absent. In addition, no stabilization of full-length Ci could be detected at the compartment boundary. However, hh-lacZ is expressed at wild-type levels in sit mutant discs, indicating that hh transcription is unaffected. This implicates Sit in the Hh pathway downstream of hh transcription and upstream of Ci stabilization (Lee, 2001).
The transcription factors Glial cells missing (Gcm) and Gcm2 are known to play a crucial role in promoting glial-cell differentiation during Drosophila embryogenesis. A central function for gcm genes has been revealed in regulating neuronal development in the postembryonic visual system. Gcm and Gcm2 are expressed in both glial and neuronal precursors within the optic lobe. Removal of gcm and gcm2 function shows that the two genes act redundantly and are required for the formation of a subset of glial cells. They also cell-autonomously control the differentiation and proliferation of specific neurons. The transcriptional regulator Dachshund acts downstream of gcm genes and is required to make lamina precursor cells and lamina neurons competent for neuronal differentiation through regulation of epidermal growth factor receptor levels. These findings further suggest that gcm genes regulate neurogenesis through collaboration with the Hedgehog-signaling pathway (Chotard, 2005).
To explore the mechanisms by which gcm genes mediate neuronal development in the optic lobe, the role of Dac was examined because its expression depends on both the activation of the Hh pathway and on gcm and gcm2 function. Genetic analysis added two findings to an understanding as to how Hh and EGF signaling work in concert to regulate neurogenesis in the lamina. It was shown that (1) dac is not required for cell divisions of LPCs and (2) that expression of dac is necessary for the upregulation and maintenance of EGF receptor expression in lamina neurons to promote their further maturation. This is consistent with findings in the developing eye imaginal disc, demonstrating that Dac promotes early progression of the morphogenetic furrow and aspects of R-cell specification but is not required for cell proliferation. In the eye, genetic interaction assays have previously established a link between Dac and EGFR signaling because dac mutant alleles were identified as suppressors of the dominant-active EGFR allele Ellipse, although the precise mechanism underlying this interaction is unclear. The current findings present evidence for one possible mechanism by demonstrating that Dac controls EGF receptor levels in the optic lobe and, in this way, makes LPCs and their progeny competent for neuronal differentiation. In Drosophila, processing of EGF ligands by Rhomboids rather than the regulation of the receptor itself has been considered to be a limiting step in EGF receptor signaling. In the rodent retina, both ligand and receptor levels have been reported to mediate different cellular responses such as proliferation and cell-fate specification. Therefore, regulating receptor levels by Dac represents an additional mechanism to modulate activity of the EGF receptor pathway in the optic lobe of flies. gcm genes can contribute to neuronal differentiation through induction of Dac. Their role in promoting mitotic divisions of LPCs, however, must involve another mechanism. Indeed, genetic analysis suggests that gcm genes regulate both developmental processes through interaction with the Hh-signaling pathway (Chotard, 2005).
That gcm genes work in concert with the Hedgehog-signaling pathway is supported by the following findings. (1) The loss-of-function phenotypes of gcm/gcm2 and hh share three characteristics because in their absence, LPCs neither enter S phase nor express the neuronal differentiation marker Dac, and show increased levels of apoptosis. (2) gcm/gcm2 loss-of-function phenotypes can be partially rescued by overexpressing activated full-length Ci in cells homozygous mutant for gcm and gcm2. One possible explanation for the partial rescue is that levels of activated Ci need to be under tight spatial and temporal control to trigger a normal cellular response. Thus, overexpressing activated Ci at high amounts compromise the ability of gcm and gcm2 homozygous mutant LPCs to express normal levels of Dac or to divide at the correct rate (Chotard, 2005).
Epistasis analysis supports a model in which gcm genes interact with the Hedgehog pathway upstream of Ci. Because loss of gcm and gcm2 function does not interfere with the general expression of Ci in LPCs, one possible mechanism is that gcm genes may indirectly affect the production of activated Ci. In the zebrafish embryo, the Zinc-finger protein Iguana/Dzip1 has recently been implicated in regulating the balance between activator and repressor forms of the vertebrate homologs of Ci, Gli1, and Gli2, possibly by modulating their nuclear activity or import. Perhaps gcm and gcm2 act in an analogous manner and regulate the production or subcellular localization of activated Ci by promoting the expression of another member of the Hh-signaling pathway. Alternatively, gcm genes may act in parallel and cooperate with Ci at the DNA level of common target genes. The dissection of the precise mechanism underlying the genetic interaction of gcm genes and the Hh pathway will require additional genetic analysis in the future (Chotard, 2005).
Gcm genes mediate neuronal differentiation in collaboration with the Hh pathway through induction of Dac. Proliferation is likely regulated by controlling a component of the cell-cycle machinery, such as Cyclin E. Indeed, in the eye and wing imaginal discs, Ci has been shown to directly promote entry into S phase by inducing increased transcription of Cyclin E. Moreover, three consensus Ci binding sites have been found within the 5' regulatory region of cyclin E (Chotard, 2005).
cubitus interruptus mutation
causes defects in every embryonic segment (Orenic, 1990). Three classes of existing mutations in the ci locus alter the regulation of ci
expression and can be used to examine ci function during development. The first class of ci
mutations causes interruptions in wing veins four and five due to inappropriate expression of the ci
product in the posterior compartment of imaginal discs. The second class of mutations eliminates CI
protein early in embryogenesis and causes the deletion of structures that are derived from the
region including and adjacent to the engrailed expressing cells. The third class of mutations
eliminates CI protein later in embryogenesis and blocks the formation of the ventral naked cuticle.
The loss of ci expression at these two different stages in embryonic development correlates with
the subsequent elimination of wingless expression (Slusarski, 1995)
The mutation ciD is mutant for two neighboring loci, cubitus interruptus and pangolin. In situ hybridication experiments with pan probes reveal that in ciD/+ heterozygote embryos, pan transcripts are detected in a pattern indistinguishable from that of ci transcripts, providing additional evidence that the molecular lesion of ciD disrupts both genes. It is a curious coincidence that pan and ci are adjacent genes, as ci encodes a transcription factor that is essential for transducing all examples of Hedgehog signaling, whereas the present evidence suggests an equivalent role for Pan in Wingless signal transduction. The fact that the ciD mutation abolishes both activities also calls for a reassessment of genetic epistasis experiments in which this allele is used to assay the relationship between Hedgehog and Wingless signaling (Brunner, 1997)
The Hedgehog (Hh) and Wingless (Wg) signaling pathways play important roles in animal development. The activities of the
two pathways depend on each other during Drosophila embryogenesis. In the embryonic segment, Wg is required in anterior
cells to sustain Hh secretion in adjacent posterior cells. In turn, Hh input is necessary for anterior cells to maintain wg
expression. The Hh and Wg pathways are mediated by the transcription factors Cubitus interruptus (Ci) and Pangolin/TCF
(Pan), respectively. Coincidentally, pan and ci are adjacent genes on the fourth chromosome in a head-to-head orientation. Genetic and in situ hybridization data indicate that ciD is a mutation affecting both ci and pan. Whereas pan is expressed ubiquitously during embryogenesis, in ciD mutant embryos pan is expressed in an intense, segmentally-repeated manner. This expression is intriguingly similar to that of the ci gene and it occurs both in homozygous and heterozygous mutant embryos. Moreover, it was found that in ciD/ciD homozyogous mutants, the typical striped expression of ci is absent; instead, ci transcripts are dispersed uniformly at low levels, like pan transcripts in wild type embryos. Molecular analysis
reveals that the ciD allele is caused by an inversion event that swaps the promoter regions and the first exons of the two
genes. The ci gene in ciD is controlled by the ubiquitous pan promoter and encodes a hybrid Ci protein that carries the
N-terminal region of Pan. The predicted Ci fusion protein product consists of the first 246 amino acids of Pan fused in frame to the Ci protein, of which the first 13 N-terminal amino acids are missing. The N-terminal domain of Pan has previously been shown to bind to the beta-catenin homolog Armadillo (Arm),
raising the possibility that Wg input, in addition to Hh input, modulates the activity of the hybrid CiD protein. Indeed, Wg signaling induces the expression of the Hh target gene patched (ptc) in ciD animals. Evidence
is provided that this effect depends on the ability of the CiD protein to bind Arm. Genetic and molecular data indicate that wild-type Pan
and CiD compete for binding to Arm, leading to a compromised transduction of the Wg signal in heterozygous ciD/+ animals
and to a dramatic enhancement of the gain-of-function activity of ciD in homozygous mutants. Thus, the Hh and the Wg
pathways are affected by the ciD mutation, and the CiD fusion protein integrates the activities of both (Schweizer, 1998).
It has been proposed that during embryogenesis ciD functions as a gain-of-function allele of ci. This is largely based on the striking finding that CiD can substute for Hh protein in driving expression of Hh-responsive genes. Specifically, in homozygous ciD mutant embryos, the ptc, wg and gooseberry genes are expressed in wider stripes, when compared to wild type, even in the absence of active Hh protein. However, homozygous ciD mutant embryos can be rescued to adult by introducing a duplication of the pan/ci genomic region (unpublished). Thus it is unlikely that the observed phenotypes can be ascribed merely to the dosage of CiD protein. Rather, it appears to be critical that no wild-type Pan protein accompanies the CiD protein in ciD homozygous embryos. This allows all Arm protein that accumulates in response to Wg to be unrestrained to bind to, and activate the CiD protein. It is predicted that the expression of an N-terminally complete Pan protein -- even if its DNA-binding activity has been impaired -- would abolish the extreme gain-of-function effect of CiD in homozygous mutant embryos. Thus it must be the simultaneous gain and loss of Arm binding sites by Ci and Pan, respectively, that confers these unusual properties to the ciD allele. Together with the observation that CiD interferes in a dominant-negative manner with Wg signaling, these results illustrate that the levels of free and accessible Arm protein critically determine the output of Wg signaling in wild-type and the output of CiD signaling in mutant situations (Schweizer, 1998 and references).
The Drosophila cubitus interruptus (ci) gene encodes a sequence-specific DNA-binding protein that
regulates transcription of Hedgehog (Hh) target genes. Activity of the Ci protein is posttranslationally
regulated by Hh signaling. In animals homozygous for the ciD mutation, however, transcription of Hh
target genes is regulated by Wingless (Wg) signaling rather than by Hh signaling. ciD is shown to
encode a chimeric protein composed of the regulatory domain of dTCF/Pangolin (Pan) and the DNA
binding domain of Ci. Pan is a Wg-regulated transcription factor that is activated by the binding of
Armadillo (Arm) to its regulatory domain. Arm is thought to activate Pan by contributing a
transactivation domain. A constitutively active form of Arm potentiates activity of a CiD
transgene and coimmunoprecipitates with CiD protein. The Wg-responsive activity of CiD could be
explained by recruitment of the Arm transactivation function to the promoters of Hh-target genes. It is
suggestrd that wild-type Ci also recruits a protein with a transactivation domain as part of its normal
mechanism of activation (Von Ohlen, 1999).
In Drosophila embryos cubitus interruptus activity is both necessary and sufficient to drive expression of HH-responsive genes, including wingless, gooseberry and patched. To demonstrate that ci is required for transduction of the HH signal, expression of wg was examined in ci null embryos when HH is ubiquitously expressed under control of a heat-shock promoter (Hs-hh). In Hs-hh embryos, wg is expressed ectopically in anteriorly expanded stripes. In ci mutants Hs-hh does not induce ectopic expression of wg. Similar results were obtained for gsb. CI is a sequence-specific DNA binding protein that drives transcription from a wingless promoter in transiently transfected cells. CI binds to the same 9 bp consensus sequence -TGGGTGGTC- as mammalian Gli and Gli3. Alteration of a single nucleotide in the core sequence prevents binding. CI activates transcription from a 5-kb fragment of the wg promoter. CI binding sites in the wg promoter are necessary for this transcriptional activation. A CI element maps to a distal 1-kb region of the 5-kb fragment. The wg promoter sequence has 10 possible Gli consensus binding sites, with three pairs of sites in the distal 1.2 kb. When putatitive CI binding sites are mutagenized, mutant fragments show a greater than 90% reduction in CI-dependent transcriptional activation. Mutagenesis of these sites completely eliminates an electrophoretic mobility shift caused by binding of CI to unmutagenized sites (Van Ohlen, 1997).
Hedgehog (HH) is an important morphogen involved in
pattern formation during Drosophila embryogenesis and
disc development. cubitus interruptus encodes a
transcription factor responsible for transducing the hh
signal in the nucleus and activating hh target gene
expression. Previous studies have shown that Ci exists in
two forms: a 75 kDa proteolytic repressor form and a 155
kDa activator form. The ratio of these forms, which is
regulated positively by hh signaling and negatively by PKA
activity, determines the on/off status of hh target gene
expression. Exogenous expression of Ci that is mutant for four
consensus PKA sites, CI(m1-4), causes ectopic expression
of wingless in vivo and a phenotype consistent with wg
overexpression. Expression of CI(m1-4), but not Ci(wt),
can rescue the hh mutant phenotype and restore wg
expression in hh mutant embryos. When PKA activity is
suppressed by expressing a dominant negative PKA
mutant, the exogenous expression of Ci(wt) results in
overexpression of wg and lethality in embryogenesis,
defects that are similar to those caused by the exogenous
expression of CI(m1-4). In addition,
in cell culture, the mutation of any one of the three serine-containing
PKA sites abolishes the proteolytic processing
of Ci. PKA is shown to directly phosphorylate
the four consensus phosphorylation sites in vitro. Taken
together, these results suggest that positive hh and negative
PKA regulation of wg gene expression converge on the
regulation of Ci phosphorylation (Y. Chen, 1999).
It can be determined whether PKA phosphorylates
consensus PKA target sites in vitro. Ci fragments of wild type Ci and of
CI(m1-4) that contain the four PKA sites (aa441-1065) were fused to
GST. Two-dimensional tryptic phosphopeptide
maps of the expressed fusion proteins were generated. There are at least 13 phosphopeptides that
are labeled by PKA in the wild-type Ci peptide. In vitro, PKA
can recognize RxS/T, the subset RRxS/T, RxxS/T and
RKxxS/T. The phosphorylation of S is preferred 40:6 over T
and in vivo, the RRxS
site is preferred 2:1 over the others. The four
consensus RRxS/T sites in Ci were chosen for mutation because they would probably
be the preferred phosphorylation sites in vivo. Scanning the
Ci fragment for all possible consensus PKA sites, it was found that all of the phosphopeptides can be accounted for by the
number of PKA consensus sites in the fusion protein. Three
of the strong spots and two weaker spots that are present in
the wild-type fragment are missing in the mutant fragment,
demonstrating that PKA can specifically and directly
phosphorylate the four RRxS/T consensus PKA sites in vitro.
The two weak spots are difficult to distinguish and may
represent only one spot or incomplete digestion of a single
peptide. GST alone was not phosphorylated (Y. Chen, 1999).
What of the positive regulation of Ci activity by hh?
Because the genetic data suggests that hh
does not regulate PKA directly, it may be that hh
affects the phosphorylation state of Ci by activating a
phosphatase, or through changing the accessibility of Ci to a
phosphatase. In support of this idea is the observation that the
phosphatase inhibitor, okadaic acid, stimulates Ci proteolysis,
even in the presence of a HH signal.
HH signaling stimulates fu kinase activity to transform full-length
Ci to a transcriptional activator. It may also be that fu activity renders full-length
Ci inaccessible to PKA phosphorylation (Y. Chen, 1999).
The manner in which Hh molecules regulate a
target cell remains poorly understood. In the Drosophila
embryo, Hh is produced in identical stripes of cells in the
posterior compartment of each segment. From these cells a
Hh signal acts in both anterior and posterior directions. In
the anterior cells, the target genes wingless and patched are
activated whereas posterior cells respond to Hh by
expressing rhomboid and patched. This study examines
the role of the transcription factor Cubitus interruptus (Ci)
in this process.
So far, Ci has been thought to be the most downstream
component of the Hh pathway, capable of activating all Hh
functions. However, the study of a null ci allele
indicates that it is actually not required for all Hh
functions. Whereas Hh and Ci are both required for
patched expression, the target genes wingless and rhomboid
have unequal requirements for Hh and Ci activity.
Hh is required for the maintenance of wingless
expression before embryonic stage 11 whereas Ci is
necessary only later during stage 11. For rhomboid
expression Hh is required positively whereas Ci exhibits
negative input. These results indicate that factors other
than Ci are necessary for Hh target gene regulation. Evidence is presented that the zinc-finger protein Teashirt is one
candidate for this activity. It is required
positively for rhomboid expression and Teashirt and Ci
act in a partially redundant manner before stage 11 to
maintain wingless expression in the trunk (Gallet, 2000).
Ci is required to transduce Hh signal in order to activate its
target genes. In cells that do not receive Hh, Ci is cleaved and
represses Hh target genes. However, compelling results point out a more complex
role for Ci activity during embryonic development of
Drosophila. The embryonic phenotype resulting
from the complete loss of Ci function is weaker than the
complete loss of Hh function. The phenotypic differences
observed between hh and ci null mutations reside in the
following observations: in ci94 embryos one observes (1) the
presence of segmentation due to maintenance of wg expression
until stage 11 and (2) the presence of denticle diversities due
to an expansion of EGF signaling illustrated by an expansion
of rho expression. Ci does not have a maternal
contribution, since ci94 homozygotes issuing from germ-line
clones homozygous for ci94, do not show a stronger phenotype
than embryos lacking only zygotic Ci product, and also embryos
hatching after ci RNA interference experiments show phenotypes similar
to ci94 embryos.
Furthermore, if rho expression present in ci mutants is due to
maternal production of Ci, one has to explain how two Ci
targets would behave differently in the absence of zygotic Ci
contribution; wg expression disappears, whereas rho is
expressed in more cells. For all these reasons it can be confidently concluded
that Ci has no maternal contribution. Consequently, other
factors are substituting for Ci activity in the transduction of Hh
signaling (Gallet, 2000).
The ci94 phenotype could be due to the fact that the ci94
mutation disrupts both activator and repressive (Cirep)
functions of Ci and that the resulting
weak phenotype could thus be due in part to the loss of Cirep
activity. Indeed, in the wing disc loss of ci gene alleviates the
repressive function of Cirep on dpp transcription; dpp is then
transcribed at a basal level.
Although Cirep could repress wg transcription, Cirep
absence in the hh;ci94 double mutant embryo is not sufficient to
induce an upregulation of wg transcription. Hence the
maintenance of wg expression until stage 11 in ci94 is not due
to the loss of Cirep activity but is controlled by another Hh type of Hh input (Gallet, 2000).
Loss of ci induces an expansion of rho expression instead of a reduction, as seen in a hh loss of function, showing that Ci is not involved in the activation of rho expression. The fact that rho disappears in tsh mutant embryos
strongly suggests that the Tsh zinc-finger protein regulates rho
expression or is at least necessary for instructing cells to
respond to Hh for rho expression.
Nevertheless, one has to explain why rho expression is
expanded in ci94 . Loss of Cirep activity could be responsible for
this effect. Indeed, overexpression of Cirep in a ci null
background or analyses of the ciCe2 mutant, which ectopically
expresses Cirep, reveals a repressive effect of Cirep on rho
expression. Therefore, Cirep could be used as a
gatekeeper in order to repress hh target genes tightly where
they should not be expressed, and thus to overcome mis-regulation
of key genes such as rho or wg. Nevertheless, these
observations contradict previous analyses showing that Cirep is
not required for correct embryogenesis, since loss of ci
function is rescued by a ci transgene lacking the Ci75 repressor
form of Ci. An alternative explanation can be gleaned from the fact
that ci94 cuticle phenotypes resemble those lacking Wg activity
during the cell specification stage. Because it has been shown that Wg exerts a
repressive role on rho expression (since absence of Wg activity
promotes ectopic expression of rho), rho expansion in ci94 could be an indirect
consequence of the late disappearance of wg expression during
stage 10-11 (Gallet, 2000).
Before stage 11, either Tsh or Ci is
sufficient for wg regulation because only the loss of both gene
activities results in a downregulation of wg, a situation
comparable with that observed in hh mutants. It is
interesting to note that Ci seems to display differential
requirements for wg maintenance and naked cuticle
differentiation in the abdomen versus the thorax. While Ci is
dispensable until stage 11 for wg expression and naked cuticle
differentiation in the abdomen, its presence in the
thorax is required. This specific Ci
function in the thorax is currently being studied (Gallet, 2000).
Both Ci and Tsh transcription factors, when overexpressed
can induce ectopic wg expression. The two factors do not display the
same features: Tsh has three atypical, widely spaced, zinc-finger
motifs, whereas Ci has conserved spacer regions
between its five zinc fingers; the binding sites identified so
far for these two proteins are different. It would be interesting
to know whether Tsh can bind directly to the wg promoter
and to identify its binding sites. It is also noteworthy that
between stage 8 and 10 wg requires, in parallel to Hh, its own
activity for the maintenance of its transcription. It has previously been shown that Tsh is a modulator of Wg signaling. Tsh becomes
phosphorylated and accumulates at a higher level in the
nucleus in Wg-receiving cells compared with cells lacking
Wg signal. Hence, in the trunk Tsh could be employed
both by Wg and Hh signaling in order to maintain wg
transcription (Gallet, 2000).
The redundancy exhibited between Tsh and Ci for wg
regulation changes after stage 10, since loss of either Ci or Tsh results
in the downregulation of wg transcripts. It is not known if this observation is the result
of a cooperation between Tsh and Ci. At least one other gene,
gooseberry, is required for the maintenance of wg transcription
at this stage, indicating that multiple inputs
for the maintenance of wg expression are necessary for normal
embryonic development (Gallet, 2000).
Studies on the developing wing blade show that Ci transduces
all Hh-delivered information. However, this study and others
on the Hh pathway support the idea that Ci is not always
involved in Hh signaling, showing that branchpoints are
common for distinct Hh signaling steps for the following five reasons. (1) It has been
shown that neither Ci nor Fused (Fu) are involved in the Hh-dependent
formation of Bolwig's organ in Drosophila. (2) A Hh-responsive wg reporter gene
with no Ci-binding sites does not require Ci activity for its
regulation until stage 11. (3) Studies on the talpid 3 gene in chicken suggest that Gli
proteins, the vertebrate homologues of Ci, regulate only a
subset of Hh target genes, the others being regulated by an
unidentified transcription factor. (4) A
Sonic hedgehog response element on the COUP-TFII
promoter binds to a factor distinct from Gli. (5) Hh signaling does not
require Ci activity to regulate rho. Although the authors favor the idea
that Tsh regulates rho expression directly in response to Hh
signal the hypothesis that Tsh plays a more
permissive role allowing Hh to regulate rho via another factor
apart from Ci cannot be excluded (Gallet, 2000 and references therein).
In conclusion, Hh requires at least two different transcription
factors during Drosophila embryogenesis to regulate its
multiple target genes and to instruct cells with precise
behaviors. The transcription factors may act independently
(e.g. Ci for ptc; Tsh for rho), cooperatively (e.g. Ci and Tsh
for wg maintenance during the cell specification phase) or
redundantly (e.g. Ci and Tsh for wg maintenance earlier during
the stabilization phase). The
possibility that other transcription factors like gooseberry might be recruited for Hh signaling cannot be excluded, especially
since denticle density is weaker in tsh;ci double mutants as
compared with hh single mutants. Furthermore
the dorsal phenotypes of the tsh;ci double mutants are weaker
than those of hh. (1) wg transcripts are still present in dorsal
patches in tsh;ci mutations whereas they are not present in hh embryos. (2) Dorsal cuticle is not as severely
perturbed in tsh;ci larvae as compared with hh
null ones (Gallet, 2000).
Finally, pathway bifurcations are involved not only at the
level of the transcription factors. The Fu kinase, which is normally required to transduce Hh signal and
to convert Ci 155 into a putative Ci act form, is not necessary in all Hh-receiving cells
during embryogenesis. While Fu is
involved anteriorly to En/Hh-expressing cells for the
maintenance of wg and ptc, it is not involved posteriorly for
the maintenance of ptc. These results correlate with the Ci
isoforms detected: anteriorly the putative Ci act form is present
but posteriorly only the Ci 155 form is detected (Gallet, 2000 and references therein).
Is the Hh pathway distally
branched? In other words, is the regulation of Ci
activity the sole output of Hh signaling? Putative Ci-independent
branches of Hh signaling were explored by
analyzing the behavior of cells that lack Ci but that had
undergone maximal activation of the Hh transduction
pathway due to the removal of Patched (Ptc). The analysis
of target gene expression and morphogenetic read-outs of
Hh in embryonic, larval and adult stages indicates that Ci
is absolutely required for all examined aspects of Hh
outputs. This is interpreted as evidence against the existence
of Ci-independent branches in the Hh signal transduction
pathway. It is proposed that most cases of apparent
Ci/Gli-independent Hh output can be attributed to the
derepression of target gene expression in the absence of
Ci/Gli repressor function (Methot, 2001).
The key result of this study is the observation that maximal
activation of the Hh pathway (i.e. complete loss of Ptc) has no
discernible effect in the absence of Ci. This is taken as evidence against a distal branching in the Hh signal transduction pathway. These results do not exclude the existence of alternative pathways between Smo and Ci, yet all these putative branches must converge at Ci. It is noted that the indispensability of Ci for Hh signaling also explains how developmental compartments are formed and
maintained. The essential difference between cells on opposite
sides of the anteroposterior compartment boundary is the
responsiveness to Hh. Posterior compartment cells do not
respond to the Hh signal, even though they are amply exposed
to Hh and appear to possess all but one of the components to
transduce Smo activity. The lack of Ci, however, precludes any
response to Hh and is thus sufficient to create a population of
cells that behaves the opposite from that of the anterior, Ci-expressing
compartment (Methot, 2001).
Although it is concluded that Hh signaling has no effect in
the absence of Ci, it is also concluded that the converse is not the
case: Ci does have a function in the absence of Hh signaling.
This can be illustrated most effectively by comparing a hh;ci
double mutant embryo with a hh single mutant one.
Although both animals completely lack the Hh signal, the
presence of a functional ci gene considerably increases the
segment polarity phenotype of hh mutants. This effect of Ci is
brought about by the default state of Ci, which is the repressor
function Ci possesses in the absence of Hh input. This function is critical for limb
development but not essential for embryogenesis. This is because an uncleavable form of Ci, CiU, can substitute for embryonic Ci in spite of the fact that it cannot form detectable amounts of Ci[rep], the repressor form of Ci. The severe phenotype of hh mutant embryos indicates that Ci[rep] activity (although not essential in a
wild-type background) can be detrimental in
circumstances where Hh signaling is abolished. This situation
is reminiscent of the Wg signal transduction pathway, where
the nuclear mediator, dTCF/Pangolin, represses Wg target
genes in the absence of Wg input. An analogous case has been
described for the Notch pathway, where the DNA-binding
factor Suppressor of Hairless has a repressive effect on
single-minded (sim) transcription in the absence of Notch
activity, yet mediates sim activation upon Notch signaling. It may be a general principle that the transcriptional targets of a signaling pathway are
repressed in the absence of the signal. Signal-mediated
induction, therefore, requires both the abolition of this
repression and the concomitant activation of transcription (Methot, 2001).
Based on this analysis, three predictions can be made regarding
the Hh pathway in other systems. (1) Loss-of-function
mutations in murine Gli genes are likely to cause phenotypes
differently from equivalent mutations in Hedgehog genes. In
particular, even a triple knockout of the Gli1, Gli2 and Gli3
genes, will presumably behave different from combined
mutations in the Sonic, Indian and Desert hedgehog genes. The
main reason for postulating this is the Hh-independent
repressor function of Gli proteins, which appears to be
primarily associated with Gli3. Lack of Shh signaling may lead
to an increase of Gli3 repressor activity, while lack of Gli3
expression has the opposite effect. Hence a double Shh Gli3
mutant may have a considerably milder phenotype than a Shh
single mutant animal (Methot, 2001).
(2) Given the conservation of the Hh transduction
pathway in different species, it is unlikely that the mammalian
Hh pathway contains end points other than Gli proteins. The
critical but genetically challenging test will be the generation
of Gli triple mutant mice and their comparison to animals
that lack in addition the Shh or the Ptc gene (Methot, 2001).
(3) These results challenge several previous studies that
claim the existence of Ci-independent outputs of the Hh
signaling pathway. Some of these studies
were conducted with a ci null allele, which removes both
activator and repressor functions of Ci. For the wing imaginal disc, lack of Ci[rep] causes the ectopic expression of certain Hh target genes. Genetic
evidence is now provided that this is also the case in embryos. It is surmised that
the seemingly Ci-independent expression of Hh-induced target
genes may reflect transcriptional derepression, owing to
removal of Ci[rep] (Methot, 2001).
The wing imaginal disc is subdivided into two nonintermingling sets of cells, the anterior (A) and posterior (P) compartments. Anterior cells require reception
of the Hedgehog (Hh) signal to segregate from P cells. Evidence is provided that Hh signaling controls A/P cell segregation not by directly modifying
structural components but by a Cubitus interruptus (Ci)-mediated transcriptional response. A shift in the balance between repressor and activator forms of Ci
toward the activator form is necessary and sufficient to define 'A-type' cell sorting behavior. Moreover, Engrailed (En), in the absence of Ci, is
sufficient to specify 'P-type' sorting. It is proposed that the opposing transcriptional activities of Ci and En control cell segregation at the A/P boundary by
regulating a single cell adhesion molecule (Dahmann, 2000).
To test the role of En and Hh-signaling components in controlling cell segregation, two experimental assays were applied. Both assays are based on the
presumption that cells maximize contact (intermingle) with cells of the same adhesiveness and minimize contact with (sort out from) cells of different
adhesiveness. In the 'round-up assay', clones of mutant cells are
assayed for their shape. Each clone is analyzed by how circular it is and how smoothly its border interfaces with surrounding tissue. The degree of roundness
of the clone and smoothness of its border is taken as a measure for the difference in adhesiveness between cells inside and outside of the clone. In the
wild-type wing imaginal disc, cell segregation is confined to the region of the compartment boundaries. Thus, in the more stringent 'choice assay,' clones
generated in the vicinity of the A/P boundary are monitored for their sorting behavior. Clones have three choices: they can (1) remain within their
compartment of origin; (2) sort completely into the territory of the adjacent compartment defining a straight border with cells of the compartment of origin at
the normal position of the A/P boundary, or (3) sort out from cells of both compartments and take up positions overlapping the normal site of the A/P
boundary. Depending on the genetic intervention, the compartment of origin of a clone was determined either by the state of the heritable and P-specific
expression of an en-lacZ reporter gene or by the position of the 'twin spot' clone, which is composed of sibling wild-type cells. The position of the A/P
boundary was inferred from the expression of a hh-lacZ reporter gene expressed exclusively in P cells (Dahmann, 2000).
Two forms of Ci are distinguished, a constitutively active form, Ci[act], and a repressive form, Ci[rep]. Autonomous and direct roles have been established for Ci[act] and En in
specifying A and P cell segregation, respectively. Evidence is also provided that Hh signaling is sufficient to specify A-type cell segregation and that it acts by
shifting the balance between Ci[rep] and Ci[act] toward low levels of Ci[rep] and high levels of Ci[act]. It is proposed that the opposing transcriptional
activities of Ci[act] and Ci[rep]/En lead to differences in the activity of a cell adhesion system at the boundary of A and P cells, thereby preventing these cell
populations from intermingling (Dahmann, 2000).
The smooth and straight boundary between compartments has been ascribed to distinct adhesive properties of cells on opposite sides of the boundary
causing these cell populations to minimize contact and sort out. In the case of the A/P boundary of the wing, one difference that
could account for the distinct sorting behavior is the exclusive presence of two transcription factors, Ci[act] and En in adjacent A and P cells, respectively.
For a long time, the view prevailed that En regulates cell segregation by autonomously and directly specifying P, as opposed to A, cell adhesiveness. This hypothesis has recently been challenged by studies indicating that En acts, at least in part, by directing the expression of
Hh and that Hh secreted by P cells induces A cells to acquire a distinct cell adhesiveness.
These studies, however, provide conflicting results as to whether or not En also has an autonomous, Hh-independent role in specifying cell segregation at the
A/P boundary. The same studies further raised, but did not address, the question of whether Hh signaling would specify cell
segregation via its normal transduction pathway by leading to a transcriptional output depending on Ci. In various other systems, the activation of signaling
receptors can lead to the posttranscriptional activation of small GTPases that can directly, without altering gene transcription, affect cytoskeletal components
and thus conceivably cell adhesion. A key tool for addressing these questions is the choice
assay. This assay allows for monitoring whether altering the activity of a gene would change a cell's compartmental preference. Using this assay,
the above questions have been addressed by systematically considering three distinct situations (Dahmann, 2000 and references therein).
Situation 1: the 'ground state,' where neither Ci nor En is present. Irrespective of their compartmental origin, clones of cells null mutant for both ci and en take up positions overlapping the normal site of the A/P boundary with smooth borders to wild-type A and P cells.
Because En is not required in A cells and because ci minus single mutant A cells behave like ci,en minus double mutant A cells, it is inferred that Ci is required in A cells for their intermingling with other A cells at the compartment boundary. Since Ci acts in these cells as a transcriptional activator, it is concluded that Hh signaling leads to a Ci-dependent transcriptional response in A cells and transcription of the immediate Hh target gene relevant for A segregation is induced, rather than repressed, in anterior
boundary cells. The behavior of ci,en minus double mutant clones also clarifies the role of En. Because clones of P cells lacking En and Ci form smooth borders with neighboring wild-type P cells that also lack Ci and, if in contact with A cells, sort partially into A territory, it is inferred that En has a function in specifying P segregation that is independent of Ci. Since Ci is required for all known responses to Hh signaling, it is concluded that En has a Hh-independent role in
determining P segregation. The observation that clones of cells mutant for both ci and en occupy A and P territory to a similar extent leads to the conclusion that Ci and En are required for most if not all aspects of the distinct segregation properties of A and P cells, and the difference between the ground state and the 'A state' brought about by Ci[act] is similar to the difference between the ground state and the 'P state' dependent on En (Dahmann, 2000).
Situation 2: Cells expressing En but lacking Ci. A more direct argument for a Ci/Hh-independent role of En in the specification of cell sorting behavior can be derived from the experiment in which anterior
clones were programmed to express low levels of En. Such cells cease to express Ci and take up positions normally occupied only by
P cells. The behavior of these cells is different from that of ground state cells that neither express Ci nor En. In
contrast to ci,en minus cells, the low level of En-expressing cells of A origin show a complete transgression to P territory, yet they do not intermingle well with P cells. This latter observation is ascribed to the unnaturally low levels of En produced in these cells (several-fold less than in wild-type P cells). These
levels may not repress ci completely and might not be sufficient to fully confer P cell adhesiveness (Dahmann, 2000).
Situation 3: Cells expressing Ci but lacking En. Posterior clones of cells expressing Ci at physiological levels, but lacking En (mutant for enE), take up positions in the territory normally only occupied by A
cells and intermingle with A cells. This behavior is dependent on Ci, since ci,en double mutant clones of P origin only partially occupy A territory and sort
out from A cells. Furthermore, overexpression of Ci in P cells leads these cells to sort out from neighboring P cells, and, if in contact with A cells, sort into A
territory. Together, by comparing situations (1) to (3), it is concluded that Ci is necessary and sufficient to specify A segregation, and, in the absence of Ci, En
is necessary and sufficient to specify P segregation (Dahmann, 2000).
Thus En has an autonomous, Hh-independent role in specifying cell segregation. In addition, Ci is necessary and
sufficient to specify A segregation. Ci is activated in anterior boundary cells by Hh whose P-specific expression is in turn controlled by En. Thus, En controls
cell segregation at the A/P boundary both by a Hh-dependent as well as a Hh-independent pathway. To determine the relative contributions of these two
pathways, situations were generated and analyzed in which En activity was altered under conditions of constant Hh signaling, or conversely, situations in which
the activity of Hh signal transduction was altered under constant En conditions. From these experiments, it is concluded that for the segregation behavior of
wing cells, the state of the Hh pathway prevails over that of En activity. This conclusion is particularly well corroborated by the finding that cells in which both
pathways are simultaneously 'on' (P cells expressing Ci), sort with A cells. The behavior of such cells may also explain why the late expression of en in
anterior boundary cells has no deleterious effects on the integrity of the compartment boundary. Like the experimental cells, these cells are
exposed to the Hh signal, coexpress ci and en, yet associate with other A cells rather than with En-expressing P cells (Dahmann, 2000).
Ci is required in A cells for proper cell segregation at the A/P boundary. Depending on the status of the Hh signaling pathway, Ci can exist in two forms with
opposing transcriptional activities (Ci[rep] and Ci[act]). These two forms of Ci regulate the expression of different subsets of
Hh target genes, some of which appear to be regulated exclusively by Ci[rep] or Ci[act]. It is argued that the A/P
sorting of wing cells is under control of both forms of Ci. This conclusion is based on findings that both Ci[rep] and Ci[act] have a profound influence on
the segregation behavior of A cells. Two observations show that Ci[rep] determines a preference for sorting into P territory. (1) A cells expressing Ci[rep] in the absence of Ci[act] or A cells overexpressing Ci[rep] in the presence of Ci[act] both take up positions occupied normally only by P cells. This is in contrast to cells
lacking Ci entirely, which take up positions overlapping the normal position of the A/P boundary. (2) P cells lacking En but expressing Ci[rep] are confined to the P compartment, unlike cells that lack En and Ci or cells that only lack En. It is inferred from this that one important function of Hh
signaling in its role of specifying A-type segregation properties is to prevent the formation of Ci[rep] in cells close to the A/P boundary (Dahmann, 2000).
The conclusion that not only prevention of Ci[rep] formation but also the induction of Ci[act] plays an important role in A/P sorting is deduced from the
observation that cells lacking both forms of Ci do not mingle with wild-type A cells expressing Ci[act] due to their vicinity to the Hh source. Moreover, the
addition of Ci to P cells, where Ci is readily converted to Ci[act], programs P cells to segregate with A cells. Because Ci[rep] influences cell segregation, one might have expected that anterior ci minus clones far away from the A/P boundary would sort out from
neighboring Ci[rep]-expressing cells. However, ci minus cells intermingle well with neighboring A cells. One likely explanation for this apparent discrepancy is the
partial derepression of hh transcription in ci mutant cells. These low Hh levels induce in neighboring cells the formation of
some Ci[act] that might neutralize remnant levels of Ci[rep]. In support of this assumption, it has been found that clones of cells double mutant for ci and hh do sort out
at anterior positions (Dahmann, 2000).
Ci and En are both DNA-binding proteins known to act as transcription factors, indicating that they control cell segregation by regulating the expression of
target genes. By analogy to dpp, a Hh target gene that is also controlled by En and both forms of Ci, a model is proposed illustrating how Ci[rep], Ci[act], and
En might shape the expression profile of a putative immediate target gene involved in cell segregation. Since in the absence of Ci and En, cells
segregate neither with A nor with P cells, they are likely expressing an intermediate level of this gene that is different from those in A or P cells.
Since Ci[rep] can control cell segregation and is present in A cells far away from the boundary, it is proposed that the basal expression of this hypothetical gene
is downregulated by Ci[rep] in these cells. In A cells close to the boundary, Hh signaling prevents the formation of Ci[rep] yet causes the formation of Ci[act],
from which it is inferred that in these cells the transcription of this target gene is upregulated. In P cells, En may repress this target gene, consistent with its role as
a transcriptional repressor. It is proposed that the opposing transcriptional activities
of Ci[act] and En lead to a large difference in the expression of this immediate target gene in cells on opposite sides of the A/P boundary (Dahmann, 2000).
In the above model, it is assumed that Ci and En control cell segregation by transcriptionally regulating one and the same gene, although it is also possible that
they regulate different genes. While at present these alternatives cannot be distinguised, the simpler model that Ci and En control the same
target gene is preferred for two reasons: (1) there is a precedent case for such a gene, dpp, which is known to be regulated by both Ci and En; (2) a difference in the expression level of a single cell adhesion molecule
(Shotgun or DE-cadherin) is sufficient for two cell populations to sort out. While it is conceivable that Ci and En directly regulate the expression of cell adhesion molecules like DE-cadherin, it is also possible that they act
more indirectly by regulating genes whose products influence the activity of uniformly expressed cell adhesion molecules. Clones of cells lacking detectable
amounts of DE-cadherin do sort out from neighboring wing disc cells; they are, however, exclusively confined to the compartment of origin,
indicating that DE-cadherin is not required for the separation of cells at the A/P boundary (Dahmann, 2000).
Why does cell segregation at the A/P boundary require two transcription factors with opposing activities? Based on the results presented here, the differential activities of
either Ci or En suffices for separating A and P cells. For Ci, this is best illustrated by the key finding that P cells forced to express Ci sort out from wild-type
P cells and segregate into A territory. Conversely, in the absence of Ci, expression of En suffices for A cells to sort into P territory. The
use of two transcription factors with opposing activities may have the advantage of increasing the fidelity of the sorting process by further contrasting the
expression levels of a common putative target gene in cells of opposite sides of the A/P boundary (Dahmann, 2000).
It seems to be a general mechanism that En controls cell segregation both in a Hh-dependent and -independent manner. In the Drosophila abdomen, En has
also been implicated to control separation of A and P cells in Hh-dependent and -independent ways. The relative contributions
of these two functions of En, however, appear to differ between the wing imaginal discs and the abdomen. While a prevalence of the Hh-dependent
pathway is found in the wing disc, the two functions of En seem to contribute equally to the separation of abdominal A and P cells. This
difference in dominance of the Hh-signal transduction pathway might be due to a more influential role of Ci[rep] in the sorting of imaginal versus abdominal cells.
It is intriguing to notice that the same intricate network that defines the strip of cells expressing Dpp also appears to restrict the activity of a putative cell
adhesion molecule to the very same cells. The use of Hh/En signaling for both setting up the Dpp organizer and segregating A and P cells may ensure that the
position and shape of the morphogen source that organizes both compartments is stably maintained during development. The prediction of a dpp-like
expression pattern provides a novel criterion for the future identification of the elusive molecules conferring cell segregation (Dahmann, 2000).
The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving
from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina
target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic
furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the
crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the
axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina
along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior
margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near
the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's
vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively.
Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The
medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Huang, 1998 and references).
Hedgehog, a secreted protein, is an inductive signal
delivered by retinal axons for the initial steps of lamina
differentiation. In the development of many tissues,
Hedgehog acts in a signal relay cascade via the induction
of secondary secreted factors. Lamina
neuronal precursors respond directly to Hedgehog signal
reception by entering S-phase, a step that is controlled by
the Hedgehog-dependent transcriptional regulator Cubitus
interruptus. The terminal differentiation of neuronal
precursors and the migration and differentiation of glia
appear to be controlled by other retinal axon-mediated
signals. Thus retinal axons impose a program of
developmental events on their postsynaptic field utilizing
distinct signals for different precursor populations (Huang, 1998).
A hallmark of Hh signal reception in many Drosophila
tissues is an increase in immunoreactivity to the C-terminal
portion of the protein Ci, a transcriptional mediator of Hh
signaling. This enhanced Ci immunoreactivity is due to inhibition of Ci proteolytic processing, a cellular
response to Hh signal reception. LPCs posterior to the lamina furrow display the enhanced Ci
immunoreactivity that would be predicted for Hh signal
reception by LPCs. In animals in which hh-
retinal axons innervate the lamina target field, cells posterior
to the lamina furrow display a level of Ci immunoreactivity
equivalent to the basal level detected anterior to the furrow, indicating that the increased Ci observed in the wild type is Hh-dependent. In smo mosaic animals, smo cells either
anterior or posterior to the lamina furrow display a basal level
of Ci immunoreactivity, while smo + cells
immediately adjacent to the portion of smo clones within the
lamina display the high Hh-dependent level (Huang, 1998).
In a number of instances, pattern formation mediated by Hh is
accompanied by cell division. The well-defined pattern of Hh-induced
cell division in the lamina provides an opportunity to
determine the point at which the Hh signal reception engages
the cell cycle machinery. LPC cell cycle progression and cell fate
determination are jointly controlled by the transcriptional regulator Cubitus interruptus.
Biochemical and epistasis experiments have placed the zinc finger molecule Ci
downstream of all other hh signaling pathway components. Ci
has been shown to bind directly to the regulatory sequences of
Hh-responsive genes. Should all Hh-mediated events of LPC maturation be found to
depend on Ci function, it could be concluded that, at least with
regard to cell proliferation and the expression of differentiation
markers, there is no branchpoint within the signaling pathway. To examine the requirement for Ci, two
recombinant constructs were used that result in either dominant Ci gain-of-function or loss-of-function phenotypes. Overexpression of the wild-type Ci gene results in a gain-of-function phenotype
that mimics activation of the Hh signaling pathway. Expression of an amino terminal fragment of Ci (hereafter
referred to as DN-Ci) results in a dominant loss-of-function
phenotype, as the normal in vivo function of this portion of the
molecule appears to be transcriptional repression of Hh target
genes. With either construct, genetically engineered ectopic expression in the lamina
region results in the expected phenotype with respect to the
lamina differentiation marker Dac. Dac expression in cells
posterior of the lamina furrow is strongly reduced or
undetectable in cells expressing DN-Ci. Conversely,
the ectopic expression of wild-type Ci results in the induction
of Dac-positive cells in the lamina target field of
animals lacking innervation from the developing eye. The effects observed with either construct
are strictly cell autonomous. Thus the results with ectopic Ci and DN-Ci expression are
consistent with the expectation that Ci modulates Hh signaling activity directly in LPCs (Huang, 1998).
cubitus interruptus:
Biological Overview
| Evolutionary Homologs
| Regulation
| Targets of Activity
| Protein Interactions
| Developmental Biology
| References
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