patched
This study addresses the question of whether the Hh pathway is distally
branched or, in other words, whether the regulation of Ci
activity is the sole output of Hh signaling. Putative Ci-independent
branches of Hh signaling are explored by
analyzing the behavior of cells that lack Ci but have
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).
High Patched levels in the wing imaginal disc, expressed in either normal or ectopic patterns, result in loss of wing vein patterning in both segmental compartments centering at the anterior-posterior border. In addition, patched inhibits the formation of the campaniform sensilla, mechanosensory neurons found in the wing blade. The patched
wing vein phenotype is modulated by mutations in hedgehog and cubitus interruptus.
patched overexpression inhibits transcription of patched and decapentaplegic and
post-transcriptionally decreases the amount of CI protein at the anterior/posterior
boundary. In wing discs, which express ectopic hedgehog, CI levels are
correspondingly elevated, suggesting that hedgehog relieves patched repression of CI
accumulation. Protein kinase A also regulates CI; protein kinase A mutant clones in the
anterior compartment have increased levels of CI protein. Thus patched influences wing
disc patterning by decreasing CI protein levels and inactivating hedgehog target genes
in the anterior compartment (Johnson, 1995).
It is believed that wing veins L3 and L4 do not respond to DPP signaling but instead L3 is determined directly by a threshold response to Hedgehog secreted across the A-P compartment boundary. It has been observed that clones of mutant patched cells in the middle of the anterior compartment are surrounded by an ectopic L3 vein which comprises wild-type cells. Similarly, loss-of-function clones of Protein kinase A, which functions like Ptc to repress ptc expression, are encircled by ectopic veins consisting of wild-type cells. Thus, cells with low levels of ptc may induce adjacent ptc+ cells to assume L3 fates. Since secreted HH is thought to be responsible for inactivating PTC, the position of the L3 primordium might be determined by a threshold response to HH diffusing from the posterior compartment (Sturtevant, 1997 and references).
The absence of ptc gene function causes a transformation of
the fate of cells in the middle part of each segment so that they form pattern elements characteristic
of cells positioned around the segment border. Mutant phenotypes show that
both segment and parasegment borders are included in the duplicated pattern of ptc mutants (Hooper, 1989).
Patched has a role in patterning in the cuticle of
the adult fly. Genetic mosaics of a lethal allele of patched show that the contribution of patched
varies in a position-specific manner. Analysis of
twin clones demonstrates that the reduced clone frequency results from a proliferation failure or cell
loss. In the region where clones upset venation, they autonomously fail to form veins and also
non-autonomously induce ectopic veins in adjacent wild-type cells. The patched transcript is present throughout the anterior
compartment, with a stripe of maximal intensity along the A/P compartment border extending into
the posterior compartment (Phillips, 1990).
A new segment polarity gene of Drosophila melanogaster, oroshigane (oro) was identified as a dominant enhancer of Bar (B). oro is a recessive embryonic lethal, and homozygous
oro embryos show variable substitution of denticles for naked cuticle. These patterns are distinctly
similar to those of hedgehog and wingless mutant embryos, which indicates that oro functions in
determining embryonic segment polarity. oro works downstream of hedgehog but upstream of dpp to enhance the Bar phenotypes. Although dpp expression is reduced in oro heterozygotes, hh expression remains the same as that found in wild-type discs. Evidence that oro function is involved in Hh signal
transduction during embryogenesis is provided by its genetic interactions with the segment polarity
genes patched and fused. ptcIN is a dominant suppressor of the oro embryonic
lethal phenotype, suggesting a close and dose-dependent relationship between oro and ptc in Hh signal
transduction. oro function is also required in imaginal development. The oro1 allele significantly reduces
decapentaplegic (but not hh) expression in the eye imaginal disc. oro enhances the
fused1 wing phenotype in a dominant manner. Based upon the interactions of oro with hh, ptc, and fu, it is
proposed that the oro gene plays important roles in Hh signal transduction (Epps, 1997).
The photoreceptors within the ommatidia of the Drosophila compound eye form a trapezoid. This
occurs in two chiral forms in the dorsal and ventral half of the eye. Ommatidia in the dorsal half of the compound eye are oriented with the R3 photoreceptor cell dorsal and anterior, the R7 photoreceptor being ventral. Ommatidia in the ventral half of the eye are inverted. This asymmetry is established during the progression of the morphogenetic furrow as it moves across the epithelium of the eye imaginal disc from posterior to anterior. As the furrow moves it lays down a new column of ommatidial clusters roughly once every 2 hours. However, the ommatidial clusters in one column are not initiated at the same moment, i.e. the first cluster is formed at the center of the furrow (the midline or future equator); subsequent clusters are formed dorsal and ventral to this at about 10-min intervals. This point at the center of the furrow is known as the firing center, an inductive node which transmits information in two directions, i.e. induction of new ommatidial columns towards the anterior and induction of new ommatidial clusters towards the dorsal and ventral poles (Reifegerste, 1997 and references).
Two manipulations were used to
induce ectopic ommatidia, in combination with molecular markers for specific positions in the retinal
field. Ectopic furrows were generated by shift of winglessl-12 homozygotes to a nonpermissive temperature for 48 hours. Loss of function patched clones were used to induce ectopic furrows, because patched functions as a negative regulator of furrow initiation. Ectopic morphogenetic furrows induced on the eye field margin (or midline) and
those induced in the body of the field have different consequences for the establishment of retinal
polarity. Ectopic clones on the midline or margin is associated with ectopic expression of early markers of retinal field polarity, while ectopic expression of clones that do not lie on the margin or midline are not associated with such markers. In cases where clones fail to induce ectopic furrows, such clones can re-specifiy polarity field markers if they lie on the margin or midline. Photoreceptor cells in the ectopic ommatidia formed by patched clones produce axons that do not always follow the normal polarity field toward the posterior and the optic stalk. In cases in which a field of ectopic ommatidial clusters is still disconnected from those formed by the endogenous field, the ectopic clusters do not find a path to the optic stalk, but converge on the center of their local field. This phenomenon may be similar to the development of axon tracts in the insect central nervous system and is consistent with a homophilic axon guidance model (Reifegerste, 1997).
An early equatorial model for retinal polarity is proposed. In this model, early events establish the dorsal/ventral polarity of the retinal field and establish the midline/equator; only later does the furrow initiate and then the firing center follows the midline, but does not form it. This idea is derived from the observation that markers of polarity are expressed in specific parts of the retinal field before furrow initiation. Thus events that initiate furrow movement on the margin or the midline re-specify the field markers, while those that lie off the margin or the midline do not. Evidence for a preexisting field of positional information comes from the characterization of the homeoprotein mirror, which seems to be involved in the establishment of retinal polarity. The gene four-jointed shows a graded expression in equatorial-polar direction along the equator in third instar eye imaginal discs. Four jointed is a putative cell surface or secreted protein. Another candidate for an equatorial signal is Wingless itself. Wingless could act early to signals from the margins inwards. A second signal from the midline could be induced by early Wingless. Mosaic clones for frizzled affect retinal polarity; these have a domineering non-autonomy on adjacent wild type tissue. Proteins similar to Frizzled have been shown to act as Wnt receptors (Reifegerste, 1997 and references).
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).
The Hh receptor Ptc, a multiple-pass membrane protein, and
the cAMP-dependent protein kinase (PKA) normally maintain
the Hh signal transduction pathway in a repressed state.
Loss-of-function mutations in either of these genes mimic Hh
signal reception and result in the cell autonomous activation of
Hh target genes in many tissues. LPCs harboring mutations for
either pka or ptc undergo differentiation cell-autonomously
and independently of retinal innervation. Mutant cells anterior to
the furrow do not differentiate precociously. This observation
is consistent with the consequences of ectopic Hh expression
in an the lamina in mutants lacking retinal innervation of the lamina.
Hh expression in regions anterior to the lamina furrow does not
induce precocious lamina differentiation, as though competence to respond to Hh is acquired by G1-phase LPCs at the anterior margin of the lamina furrow. Within the lamina
target field, wild-type cells neighboring the pka or ptc
mutant cells are never observed to express Dac. Thus
activation of the Hh pathway by loss-of-function in either gene
results in a strictly autonomous induction of LPC maturation.
These results permit the conclusion that the terminal cell
division and differentiation of LPCs both require the direct reception of the Hh signal (Huang, 1998).
Like the Drosophila embryo, the abdomen of the adult
consists of alternating anterior (A) and posterior (P)
compartments. However the wing is made by only part of
one A and part of one P compartment. The abdomen
therefore offers an opportunity to compare two
compartment borders (A/P is within the segment and P/A
intervenes between two segments), and ask if they act
differently in pattern formation. In the embryo, abdomen
and wing P compartment cells express the selector gene
engrailed and secrete Hedgehog protein while A
compartment cells need the patched and smoothened genes
in order to respond to Hedgehog. Clones of cells were produced
with altered activities for the engrailed, patched and
smoothened genes. The results confirm (1) that the state of
engrailed, whether 'off' or 'on', determines whether a cell
is A or P type and (2) that Hedgehog signaling, coming
from the adjacent P compartments across both A/P and
P/A boundaries, organizes the patterning of all the A cells.
Four new aspects of compartments
and the expression of engrailed in the abdomen have been uncovered. (1) engrailed
acts in the A compartment: Hedgehog leaves the P cells and
crosses the A/P boundary where it induces engrailed in a
narrow band of A cells. engrailed causes these cells to form
a special type of cuticle. No similar effect occurs when
Hedgehog crosses the P/A border. (2) The
polarity changes induced by the clones were examined, and a
working hypothesis was generated, as follows: polarity is organized, in both
compartments, by molecule(s) emanating from the A/P but
not the P/A boundaries. (3) It has been shown that both the A
and P compartments are each divided into anterior and
posterior subdomains. This additional stratification makes
the A/P and the P/A boundaries fundamentally distinct
from one another. (4) When engrailed is
removed from the P cells (of segment A5, for example) the P cells transform
not into A cells of the same segment, but into A cells of the
same parasegment (segment A6) (Lawrence, 1999).
The cells of the dorsal epidermis of the adult abdomen in
Drosophila exhibit two properties: (1) a scalar property,
shown by the identity of the cuticle they secrete, and (2)
a vectorial property, indicated by the orientation of hairs and
bristles. The scalar properties are represented by the presence of subdomains within both the A and P
compartments. ptc-;en- cells at the front and the back of
the A compartment give different transformations, confirming that
there are two domains in A. These domains correspond largely to the territories of a1,
a2 (no bristles) and a3, a4, a5 cuticle (with bristles). Removal of the Notch (N) gene from these two regions
gives different outcomes: N- clones in a2 cuticle make epidermal cells,
while those in a3 do not. It follows that
the cells composing a2 (non-neurogenic) and a3 (neurogenic) are fundamentally distinct. The P compartment is also subdivided.
Thus, the loss of en from posterior P cells converts them from
making p1 cuticle to either a1 or a2, depending on whether they
can receive the Hh signal. The removal of en from anterior P
cells causes them to make either a5 or a3 cuticle, again
depending on whether they can receive Hh (Lawrence, 1999).
Why should there be such a subdivision of the
compartments? Perhaps it is connected with making a
distinction between A/P and the P/A borders, for if both were
simply an interface between A and P cells, they would differ
only in their orientation. It is not known
what agent discriminates between the two domains in either
compartment; perhaps one regulatory gene would be sufficient
for both: its expression could flank the segment boundary,
redefining nearby regions of the A and P compartments.
The domains are not maintained by cell
lineage. Analogous domains are found in the legs, where A
compartment cells respond to Hh by expressing high levels of
either Decapentaplegic or Wingless, depending on
whether they are located dorsally or ventrally in the appendage. This dorsoventral bias in response is
established early in development, and then maintained, not by
lineage, but by feedback between Wg- and Dpp-secreting cells (Lawrence, 1999).
The vector property of the epidermis is represented by the orientation of adult hairs. A model has been proposed where Hh crosses over from P to
A and elicits production of a `diffusible Factor X' that grades
away anteriorly from the A/P border, and has a long range; the
cells are oriented by the vector of this gradient. For simplicity, this discussion will be restricted to the
posterior domain of the A compartments. The A/P
boundaries cannot be unique sources of X, for polarity changes
also occur when cells from one level of A confront those from
another (e.g. when a5 and a3 cells meet at the edge of ptc-;en-
clones). This suggests that away from the compartment
boundaries, cells also produce X, the quantity depending on the
amount of Hh received. It is therefore imagined that a gradient of
X would be formed both by the graded production of X (high
near the A/P boundary, low further away) and also by its further
spread into territory (a3) where Hh is low or absent. Note that this model fits with most of the results for it makes
the A/P boundaries the organisers: whenever
ectopic A/P boundaries are generated by the clones, their
orientation correlates with the polarity of territory nearby; this
is most clearly seen at the back of en-expressing clones. The line where polarity switches from
normal to reversed does not occur at a fixed position in the
segment but rather appears to be related to the
position of nearby A/P borders (Lawrence, 1999).
en- clones in the P compartment make A cuticle. In the anterior
part of P these clones have normal polarity. In the posterior part
of P the whole clone displays reversed polarity, as do some
cells outside the clone. In order to understand this (at least, in part),
consider the behaviour of ptc- clones in the A compartment:
they behave differently depending on their distance from the
A/P border, the presumed source of X. At the back of the A
compartment they are near that border and have little or no
effect on polarity, but when closer to the front of A, they
repolarize several rows of cells in the surround. This is explained
as follows: near the source of X, where the ambient level is
high, limited production of X might not much affect the
concentration landscape. But far from the source, where the
local concentration of X would be low, any effects would appear greater.
Likewise, if there were a polarizing factor similar to X in the
P compartment, then clones of en minus cells that produce complete
or partial borders might become ectopic sources of this factor:
they would produce altered polarities only in an environment
where the level of the factor were low. This argument suggests
that a polarising factor 'Y' for the P compartment might emanate
from the A/P border and spread backward. Thus the evidence
is consistent with the idea that polarizing signals spread in both
directions from the A/P boundaries. The P/A (segment)
boundaries might act to stop these factors trespassing into the
next segment, just as they appear to block the movement of
Wingless protein (Lawrence, 1999).
The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).
It has been concluded that Hh acts indirectly via another system (a gradient of 'X') to effect polarity. The evidence was based on clones that lacked such downstream genes as patched (ptc) or cAMP-dependent protein kinase 1 (Pka). In the A compartments, Ptc and Pka proteins act within cells to prevent the Hh pathway from being activated inappropriately; if either protein is removed the Hh pathway becomes constitutively activated within the mutant cells themselves. With respect to the type of cuticle (the scalar output of Hh) the results fit the model; the mutant cells make the cuticle normally made by cells responding strongly to Hedgehog and all the cells outside the clone make the normal type of cuticle (a cell-autonomous effect). However, with respect to polarity (the vectorial output of Hh), the results are different; polarity is altered in the wild-type cells up to several cell diameters away from the clone (a cell non-autonomous effect). Although it has been argued that these effects were not due to Hh itself, the possibility was not eliminated that low levels of ectopic Hh might be produced by the clone and diffuse out, being sufficient to repolarize the cells without changing the scalar. This study now disproves this possibility by making clones that lack both effective Ptc protein and the hh gene. These clones still cause repolarization in the back half of the clone and behind it arguing strongly that the Hh protein is a component of 'X' and raising again the question, what is X? X should be engendered downstream of Hh receipt, which is where the search is started (Lawrence, 2002).
If the production of X depends at least in part on omb, then ptc- clones, in which the Hh pathway has been constitutively activated, should produce little or no X if they also lack omb. Clones were made that were both ptc- and omb-; these clones form a6 cuticle as do ptc- clones. However, in the middle of the A compartment and unlike ptc- clones in that position, they fail to repolarize behind, but reverse their polarity in front -- as do omb- cells. Similarly, omb- ptc- clones situated at the back of the A compartment behave like omb- clones, the whole being reversed in polarity (and not like ptc- clones in the same location, that have normal polarity). Thus in terms of the type of the cuticle (the scalar), omb- ptc- behave as ptc- clones, but in terms of the vector they behave as omb- clones. These results confirm that Hh induces X production through the action of omb (Lawrence, 2002).
The model for X suggests that, if omb were ectopically activated in cells at the front of the A compartment, those cells could become a source of X. Indeed omb-expressing clones can repolarize the cells behind them -- as if there were a local peak in the X distribution (Lawrence, 2002).
smoothened (smo), is an essential component of Hh transduction; without it the cells cannot see Hh protein. As regards polarity, one would expect neither omb- nor smo- clones to produce X and for their phenotype to be the same. Although this is generally the case, the effects of smo- and omb- differ for clones located at the back of the A compartment. Polarity within these omb- clones is completely reversed, consistent with the model, whereas the corresponding smo- clones are reversed only within the anterior portion of the clone, polarity returning to normal at the very back of the A compartment. The preferred explanation for this discrepancy is that Smo protein perdures in smo- clones, allowing partial rescue of the smo mutant phenotype, particularly at the back of the A compartment, where Hh is most abundant. This rescue could allow production of X, enough to restore normal polarity at the back of the clone, but not enough to specify a4 cuticle or to upregulate ptc.lacZ. For both smo- and omb- clones, some Hh would be expected to move forward across the clone and induce an ectopic peak of X production in more anterior, wild-type cells, accounting for the polarity reversals that are observed in both cases (Lawrence, 2002).
To test this explanation Hh receipt was blocked by a different method that is not so subject to perdurance: a marked clone was made that contained no wild-type Ptc, but provided instead a mutant form of Ptc that is ineffective at transducing the Hh signal. Such clones behave like smo- clones in most respects, including making a3 cuticle instead of a4, a5 or a6 cuticle in the back half of the A compartment, and causing polarity reversals both within and anterior to the clone. However, unlike smo- clones, the polarity at the back of these clones does not return to normal. Instead, in the majority of cases, polarity remains reversed all the way to the back edge of the clone, and sometimes beyond, as observed for omb- clones in the same position. These results support the perdurance explanation for the smo- clones and are consistent with the working model, which is based mainly on the results with omb (Lawrence, 2002).
The Hedgehog (Hh) signal has an inductive role during Drosophila development. Patched is part of the Hedgehog-receptor
complex and shows a repressive function on the signaling cascade, which is alleviated in the presence of Hh. The first dominant gain-of-function allele of patched has been identified: Confused (patchedCon). Analysis of the patchedCon allele has uncovered novel features of the reception and function of the Hh signal. At least three different regions of gene expression
were identified and a gradient of cell affinities was established in response to Hh. A new state of Cubitus interruptus activity,
responsible for the activation of araucan and caupolican genes of the iroquois complex, is described. This state has been shown to be independent of Fused kinase
function. In the disc, patchedCon behaves like fused mutants and can be rescued by Suppressor of fused mutations.
However, fused mutants are embryonic lethal while patchedCon is not, suggesting that Patched could interpret Hedgehog
signaling differently in the embryo and in the adult (Muller, 2000).
Thus ptcCon has partially impaired Hh-signaling
transduction, interpreting the surrounding Hh concentration
that reaches the cell as lower than it really is. Changes
in Hh concentration alter Hh target gene expression in
ptcCon cells and, subsequently, the ptcCon phenotype, indicating that ptcCon affects the interpretation of Hh levels. The lesion of the ptcCon protein is located in the first
extracellular loop of the Ptc protein, which, in vertebrates,
is involved in binding Shh. A putative explanation for this
would be that ptcCon binds Hh less efficiently, impeding the
proper transduction of the signal. The
transduction of the Hh signal can be interpreted as a balance between Ptc
protein interacting with Hh to open the pathway and Ptc
protein interacting somehow with Smo to block the pathway.
The interaction between Ptc and Hh and between Ptc
and Smo could take place inside the cell in distinct subcellular
compartments. Hh could sequester Ptc to avoid the
negative, direct or indirect, interaction with Smo. If this
were the situation, given that ptcCon binds Hh less efficiently, the result would be more Ptc protein interacting with Smo. The
increase in Ptc-Smo interaction could impede the release or
modification of Smo to transduce the signal. This explanation
also accounts for the dominant effect of ptcCon. In a
heterozygotic fly, both forms of Ptc would be present. One
of them, ptcCon, would have less affinity for Hh, which
would reduce the reception of Hh at the A-P border. Thus,
A cells would receive less Hh because ptcCon competes with
the wild-type protein for the reception of Hh. The high Hh
levels that induce some responses such as anterior En
expression would not be read, provoking the dominant
phenotype of ptcCon (Muller, 2000).
Depending on the domain where a ptcCon clone is
located, the results of blocking the Hh signal are different. The specification of vein 3 has been a subject of debate
due to its morphogenetic implications. Some lines of evidence
suggest that vein 3 differentiation depends upon the
presence of high levels of Dpp. Nevertheless, ectopic expression of Dpp does
not affect vein 3 or promote differentiation in a genetic
background in which Hh signaling is impaired. In ptcCon and fu clones, dpp
is not expressed and yet both types of clones differentiate
vein 3 when the Hh concentration is sufficient to
induce a response. When a dose of hh is removed, ptcCon mutant cells do not differentiate vein 3. It follows that Hh,
and not Dpp, specifies the location of vein 3, and Dpp has a
permissive role in establishing a broad, competent domain
for vein 3 differentiation. The results presented here confirm
that Hh forms a concentration gradient in the A
compartment and strongly suggest that Hh acts as a morphogen in the wing disc to pattern the central region of the wing (Muller, 2000).
In the abdomen, most morphogenetic functions are mediated
by Hh, and although other morphogenetic molecules
might exist, Dpp does not seem to have a role in patterning
the abdomen. In ptcCon discs, dpp is not expressed and this may account for the lack of growth in these discs. Nevertheless, the larvae
reach the third larval instar stage and the discs are similar in size
to those from the second larval instar. Thus, Dpp activation
in response to Hh seemed to function only after the second
larval instar to promote growth and patterning of the discs. Hh may have evolved as the primary morphogen of adult structures and it was not until the advent of appendages during evolution that Dpp was recruited for long-range
patterning of structures. This may be due to a need for a
higher diffusion capacity to pattern the new structures
(wings, antennae, and legs) (Muller, 2000).
Hh is also responsible for inducing a change in cell
affinity. Lack of Smo completely abolishes Hh signaling
and, consequently, impedes the change in A-cell affinity. Although the involvement of Hh and Smo in this process has been clear, that of the Hh-receptor Ptc has
not. There is the possibility of a second signaling pathway,
dependent on Smo but not on Ptc, which would mediate the
responses for changing cell affinity. In this study it is concluded that
the establishment of the lineage restriction border (LRB) depends upon the correct Ptc perception of the Hh signal. The mechanism by which the LRB arises raises a further question: why do A cells responding to Hh not form a
restriction border with A cells not responding to Hh? ptcCon clones close to the P compartment present straight
boundaries with both A and P cells, indicating that the cell
affinity of ptcCon cells is different from that of both
populations of cells. In ptcCon cells, there is a weak
response to Hh, which may be responsible for a discrete
change in cell affinities in ptcCon cells, making them different
from both the P cells, which do not respond to Hh, and
the adjacent A cells, which do respond to Hh. When a copy of hh is removed, ptcCon clones take up more posterior
positions and adopted more wiggly boundaries with P cells,
indicating that their cell affinity is more similar to that of
P cells. Changes in cell affinities seem to form in a gradient
fashion, with different changes in response to different
concentrations of Hh. Adjacent A cells receiving the Hh
signal may have such similar cell affinities that no restriction
border forms between A cells. A similar mechanism
has been suggested to occur in the abdomen of Drosophila (Muller, 2000).
In ptcCon clones, a unique experimental situation is presented
in that reception of Hh signaling is severely impaired,
allowing the accumulation of Ci in the cytoplasm without
the activation of dpp. ptcCon
clones in the wing differentiate vein 3 when close to the P compartment and substitute vein 4 for vein 3. This is in accordance with the
activation of Caup in ptcCon
clones, which is involved in determining vein 3 in the wing
imaginal disc. When lowering the concentration of Hh by
removing a copy of hh, vein 3 is not induced in ptcCon
clones and the levels of cytoplasmic Ci are low,
similar to smo clones that do not differentiate vein 3. In the same line, ptcCon clones close to but not touching the A-P border do not
develop vein 3 nor express Caup. Since ptcCon
cells interpret high Hh levels as low, these
results ascribe the role of determining the position and
differentiation of vein 3 to low levels of Hh. Furthermore,
Ci accumulation in the cytoplasm indicates the activation
of Ci to induce expression of Caup and differentiation of vein 3 (Muller, 2000).
The fact that ptcCon imaginal discs reach second larval
instar suggests that it is not until this stage that the
responses to Hh affected by ptcCon are needed. However,
there is still a paradox: if fu clones behave like ptcCon clones, why are smo and fu mutants embryonic lethal while ptcCon is not? It is proposed that ptcCon affects a function of Ptc that is needed only in larval stages, perhaps to interact with another protein, providing further refinements to Hh-signaling
interpretation. Alternatively, in the embryo, another
protein may participate in the Hh-receptor complex
(so far formed by Ptc and Smo) by binding to Ptc through a
domain not affected by the ptcCon mutation. Evidence for
the existence of other proteins involved in receiving the Hh
signal is provided by the embryonic ptc;hh double-mutant
phenotype, which is not identical to that of ptc, indicating
that Ptc alone does not receive the Hh signal in the embryo. A putative candidate, Hip, has been recently found in vertebrates. Hip is a membrane protein that binds Hh with the same affinity as that of Ptc and, similar to Ptc, is
expressed and modified by Hh. Since ptcCon would affect a
domain of Ptc needed only in larval stages, Ptc function in
embryos would be unaltered (Muller, 2000).
Ci is involved in controlling the transcription of Hh
target genes. It has been recently proposed that Hh controls
both the repressing and the activating functions of Ci. Apart
from negatively regulating the generation of a repressor
form of Ci (Ci-75), Hh controls the activation of Ci. Only two forms of
Ci are detected in a Western blot: a 75-kDa form which bears repressor activity and a 155-kDa form which seems to act as an activator. Two activation states for Ci have been described, both of which are probably modifications of the Ci-155 form.
One is responsible for inducing en and the other for inducing ptc and dpp. Neither of these responses is produced in the absence of fu or in ptcCon cells (Muller, 2000).
The unmasking of a third level of apparent
Ci activity is reported that is independent of the other two levels. This new state of Ci activity is responsible for the
activation of iro and the differentiation of vein 3 in the
wing. The other two levels of Ci activity arise from high
levels of Hh and depend on Fu activity. The new state of Ci
is activated by low levels of Hh and is Fu independent.
Thus, Hh signaling activates two different pathways
through inhibition of Ptc function. Fu would be involved in
mediating transduction of the signal in one of these pathways.
The second pathway would modify Ci to activate it in
a Fu-independent manner. It has been suggested that low levels of Hh activate a
new form of Ci, named 'Ci default', which does not depend
on Fu activity (Muller, 2000).
The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. A genetic screen based on the FRT/FLP system has been to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source, 250,000 mutagenized chromosomes were screened. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, a class of patched (ptc) alleles was identified exhibiting a novel phenotype. These results demonstrate the value of this setup in the identification of genes involved in distinct wing-patterning processes (Végh, 2003).
A total of 250,000 mutant chromosomes covering the X chromosome and both major autosomes were screened. Four complementation groups were identified that affected wing patterning similar to mutations in smo. The largest of these groups represents alleles in smo itself. Two groups exhibiting a subset of smo phenotypes represent new alleles of fused and collier/knot. Fused is a positive regulator of Hh signaling, and collier/knot is an Hh target gene required for the formation of the L3/L4 intervein region. Surprisingly, the remaining complementation group turned out to consist of novel ptc alleles with striking characteristics. Molecularly, they represent point mutations causing an amino acid substitution in either the first or the second large extracellular loop. In contrast to ptc null alleles, homozygous mutant clones failed to upregulate Hh target genes even in the presence of Hh. Together these findings suggest that the mutant proteins repress Smo constitutively, most likely because they fail to bind Hh. Animals mutant for trans-heterozygous combinations of these new ptc alleles with ptcS2 are fully viable. The ptcS2 product lacks the ability to repress Smo but is able to sequester, and hence bind to, Hh. The intragenic complementation that was observed suggests that both functions of Ptc, binding of Hh and repression of Smo, can be provided by individual proteins that possess only one of each. Recently, it was shown that a combination of two proteins, one consisting of the N- and the other the C-terminal half of Ptc, reconstitutes Ptc function. Although these experiments cannot be directly compared with the findings in this study, together they do suggest that Ptc function can be separated intramolecularly into independent modules of N- vs. C-terminal and extra- vs. intracellular domains. One possible scenario that could explain the intragenic complementation would be if Ptc proteins act in a multimeric complex (Végh, 2003).
The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. Endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and both Hh and Ptc are targeted to lysosomal degradation. The ptc14 mutant does not block Hh spreading, because it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as does the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, both in this mutant and in those not producing Ptc protein, Hh still occurs in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization (Torrioja, 2004).
Through the analysis of ptc14 (a mutant that does not internalize Hh but is able to perform Hh signal transduction) this study shows that both proposed Ptc functions are genetically uncoupled. Ptc limits the Hh gradient by internalizing Hh in a dynamin-dependent manner, and this Hh-Ptc complex is targeted to the degradation pathway. These findings strongly suggest that internalization mediated by Ptc shapes the Hh gradient and also leads to the challenging suggestion that Hh signaling can occur in the absence of Ptc-mediated Hh internalization. The two functions of Ptc in Hh signal transduction are discussed in the light of these results (Torrioja, 2004).
Hh and Ptc sorting to the endocytic membrane-bound
compartment plays a crucial role in modulating Hh levels during development. A strong support of the conclusions in this work comes from the analysis of the ptc14 allele. Although ptc14 mutants are lethal with a strong ptc- embryonic phenotype, ptc14 mutant cells in the imaginal discs show an effect only when the clone touches the AP compartment border but not in any other part of the disc. This result indicates that the presence of Hh is required to reveal a defect in Ptc14 function. This Hh requirement has been probed by the lack of activation of the Hh targets in ptc14 cells in the absence of Hh, either in the embryos or in the imaginal discs. The complementation of ptc14 with ptcS2 allele, which is considered as null for blocking Hh signal transduction and acts as dominant negative, indicates that Ptc14 does not have a greater sensibility to Hh than the Ptc wild-type protein. Conversely, it has been shown in this study that there is a decrease of internalization of Hh in ptc14 mutant clones compared with wild-type Ptc territory and an extension of the
range of Hh gradient. Therefore, it can be concluded that Ptc14 is unable to sequester Hh efficiently in either the embryo or imaginal discs and that the ptc14 embryonic phenotype would be the result of greater spreading of Hh and not due to the constitutive activation of the Hh pathway (Torrioja, 2004).
Ptc14 responds to Hh as does the wild-type Ptc protein and activates the signaling pathway indicating that the interaction of Ptc14 and
Hh is probably normal. However, this Hh-Ptc interaction does not necessarily imply sequestration. Although Ptc14 occurs at the plasma membrane, no internalization of Hh or extracellular Hh accumulation occurs in ptc14 mutant clones. These results, therefore, suggest that Hh-Ptc interaction is not sufficient to sequester Hh and that an active internalization process of Hh mediated by Ptc to control Hh gradient is required. This Hh internalization mediated by Ptc is Dynamin-dependent, based on the membrane accumulation of Hh and Ptc in shi mutant clones and the lack of accumulation of Hh in shits1; ptc16 double mutant clones. However, the initiation of the internalization process is not blocked in shi mutants because Dynamin is required for fission of clathrin-coated vesicles after the internalization process has already started. This fact would explain why Hh gradient and signaling is
not extended when endocytosis is blocked in shi mutant cells. Since
Ptc14 seems to have a problem in entering the endocytic compartment and no Hh accumulation is found in shits1;
ptc14 double mutant clones, it is concluded that the initiation of the internalization process does not occur in Ptc14. Taken together, these data indicate that only when Ptc forces Hh to the endocytic
pathway Hh is sequestered in the receiving cells (Torrioja, 2004).
To block the degradative pathway, deep orange (dor) mutants were used. dor, one of the mutations that affects eye pigmentation in Drosophila, is required for normal delivery of proteins to lysosomes. The behavior of Hh and Ptc in dor- cells indicates that
after sequestration, Ptc internalizes Hh, and both Hh and Ptc are degraded.
Thus, controlling both endocytosis and degradation of Hh modulates its
gradient. Similar mechanisms have been described for controlling the
asymmetric gradient of Wg in embryonic segments. It is
possible that additional factors may contribute to shaping the Hh gradient, because in large ptc- clones close to the AP border, which lack Ptc protein to sequester Hh, an Hh gradient in endocytic vesicles is
also observed, although the range of this gradient is more extended than in
wild-type cells. This is consistent with two mechanisms of Hh
internalization in Hh receiving, one mediated by Ptc
and another not mediated by Ptc (Torrioja, 2004).
From studies in both vertebrates and Drosophila, it was thought
that Hh protein binds to Ptc. Ptc is then internalized and traffics Hh to endosomal compartments where both are degraded, the
entire process triggering activation of the Hh pathway. It is shown in this study that Ptc14 responds to Hh as would the wild-type Ptc protein in activating the pathway. However, Ptc14 does not internalize Hh to the endocytic compartment because it is defective in endocytosis. It is therefore suggested that the massive Hh internalization by Ptc to control the gradient is
not a requirement for Hh pathway signal transduction (Torrioja, 2004).
In Hh signal transduction, the cellular mechanisms that regulate Smo
function remain unclear, although the distribution of Ptc/Smo suggests that Ptc destabilizes Smo levels. It has also been proposed that Ptc-mediated Hh internalization changes the subcellular localization of Ptc preventing Smo downregulation. Furthermore, in cultured cells, Shh induces the segregation of Ptc and Smo in endosomes, allowing Smo signaling, independently of Ptc. It is known, however, that binding of Shh to Ptc is not sufficient to relieve the repression of the Hh pathway (Torrioja, 2004).
As in wild-type cells, in the absence of Hh, Ptc14
downregulates both Smo levels and Smo activity, while in the presence of Hh, the normal upregulation of Smo occurs. Consequently, Ptc14 levels are high at the AP border because upregulation of Ptc by Hh occurs in the absence of internalization of Hh to the degradative pathway. It might then be expected that the high levels of Ptc14 not targeted to the degradative pathway would block Smo activity. However, against all predictions, the presence of Hh is still able to release Smo activity in mutant ptc14 cells. Thus, there must be a positive mediator of Smo activity to overcome the repressive effect of Ptc14 and allow Hh pathway activation in response to Hh. Alternatively, if Ptc14 is located at the plasma membrane, it could control Smo activity without entering the endocytic compartment by regulating the entrance of small molecules, as has been proposed. In
fact, Ptc is similar to a family of bacterial proton-gradient-driven
transmembrane molecule transporters known as RND proteins.
Accordingly, as a membrane transporter, Ptc could indirectly inhibit Smo
through translocation of a small molecule that conformationally regulates the active state of Smo. The inter-allelic complementation of Ptc suggests that Ptc has the oligomeric structure needed for this type of transporter (Torrioja, 2004).
Although one of the normal functions of Ptc is to mediate Hh
internalization, the data demonstrate the presence of internalized Hh vesicles in the absence of Ptc protein. It is therefore suggested that another receptor mediates Hh internalization in Hh-receiving cells. This molecule could act as a positive mediator of Hh signaling. Several observations have been published
that cannot easily be reconciled with the idea of Ptc acting as the only
receptor for Hh. For example, it was found that Hh activates signal
transduction in both A and P compartment cells of wing imaginal discs, despite the absence of Ptc in P cells. Furthermore, it has been reported that some neuroblasts in Drosophila embryos, the maturation of which is dependent on Hh, do not express or require
Ptc. This suggests that a receptor other than Ptc mediates Hh signaling. Recently, the glypican protein Dally-like, which belongs to the heparan sulfate proteoglycan protein family, was found to be required for Hh signal transduction and probably for the reception of the Hh signal in
Drosophila tissue culture cells. Dally-like
could act as co-receptor for Hh and it would be interesting to know if
Dally-like is required for Hh endocytosis. In addition, the large glycoprotein 'Megalin' has recently been identified as a Shh-binding protein. Megalin is a multi-ligand-binding protein of the low-density lipoprotein (LDL) receptor family whose function is to mediate the endocytosis of ligands. The finding that megalin-mediated endocytosed N-Shh is not efficiently targeted to lysosomes for degradation suggests that N-Shh may also traffic in complexes with Megalin and thus be recycled and/or transcytosed. In the Wg pathway, specific LDL receptor-related proteins are essential co-receptors for Wnt ligands. Further investigation will determine whether LDL receptor-related proteins could function as co-receptors that internalize Hh in the absence of Ptc. Alternatively, these proteins could be required for endocytosis and further delivery of Hh to Ptc in intracellular vesicles,
perhaps facilitating the transcytosis of Hh. A future challenge will be to
find other molecules that internalize Hh and to understand how Hh interacts with Smo to activate the Hh pathway (Torrioja, 2004).
The localized expression of Hedgehog (Hh) at the extreme
anterior of Drosophila ovarioles suggests that it might
provide an asymmetric cue that patterns developing egg
chambers along the anteroposterior axis. Ectopic or
excessive Hh signaling disrupts egg chamber patterning
dramatically through primary effects at two developmental
stages. (1) Excess Hh signaling in somatic stem cells
stimulates somatic cell over-proliferation. This likely
disrupts the earliest interactions between somatic and
germline cells and may account for the frequent mis-positioning
of oocytes within egg chambers. (2) The
initiation of the developmental programs of follicle cell
lineages appears to be delayed by ectopic Hh signaling. This
may account for the formation of ectopic polar cells, the
extended proliferation of follicle cells and the defective
differentiation of posterior follicle cells, which, in turn,
disrupts polarity within the oocyte. Somatic cells in the
ovary cannot proliferate normally in the absence of Hh or
Smoothened activity. Loss of protein kinase A activity
restores the proliferation of somatic cells in the absence of
Hh activity and allows the formation of normally patterned
ovarioles. Hence, localized Hh is not essential to direct egg
chamber patterning (Zhang, 2000).
Hh signaling in Drosophila generally regulates the abundance
and activity of Ci proteins without altering CI mRNA levels. By contrast, vertebrate Hh homologs
frequently regulate transcription of the Ci-related GLI family
of transcriptional effectors. The induction
of CI RNA in ptc mutant follicle cells provides the first evidence
that this circuitry can also be found in Drosophila.
Other consequences of altering the activity of Hh signaling
components in ovarian somatic cells substantiate the
hypothesis that Hh signaling activates at least two distinct
intracellular pathways. One pathway, involving protection of Ci-155
from proteolysis and perhaps also release from
cytoplasmic anchoring, is phenocopied by PKA and cos2
mutations. In the ovary, cos2 mutations elicit stronger
phenotypes than PKA mutations, perhaps because cos2
mutations preferentially disrupt cytoplasmic anchoring of Ci-155.
The second pathway increases the specific activity of Ci-155
in opposition to the inhibitory effects of Su(fu). This pathway is elicited by ptc, but not
by PKA mutations and requires Fu kinase activity. In
accordance with this model, PKA Su(fu) double mutant cells
produce phenotypes almost as strong as for ptc mutants in
ovaries, whereas ptc fu double mutant cells exhibit minimal
phenotypes and PKA mutant phenotypes are not greatly
altered by additional loss of Fu kinase activity.
In imaginal discs high level Hh signaling to nearby cells is
phenocopied by ptc mutations and requires Fu kinase activity,
whereas only low level Hh signaling to more distant cells can
be phenocopied by PKA mutations and does not require Fu
kinase activity. PKA mutations in somatic
ovarian cells can effectively substitute for Hh activity: Fu
kinase activity is not essential for somatic cell proliferation and ptc mutations engender excessive
Hh signaling phenotypes even in the absence of Hh activity.
Hence, it is surmised that ovarian somatic cells normally undergo
only low levels of Hh signaling, in keeping with the
observation that the source of Hh in the germarium is separated
from its target cells by several cell diameters (Zhang, 2000).
The rescue of apparently normal oogenesis in hhts animals at
the restrictive temperature by PKA mutations in somatic stem
cells implies that there is no essential role for spatially graded
Hh levels in the germarium. However, the level of Hh signaling
must fall within certain bounds for oogenesis to proceed
normally. Normal rates of somatic cell proliferation require
some Hh signaling but also require that Ptc limits Hh signaling. Ptc must also restrain Hh signaling in
order to allow somatic cells to enter the developmental
program appropriate to their lineage in a timely fashion.
It is not clear at this stage whether Hh signaling has any
essential function in oogenesis other than stimulating cell
proliferation. In one case, normal egg chambers can include
smo mutant cells in a variety of positions. In particular, polar
cells can form in normal numbers and at the correct position
from within a group of smo mutant cells, which are presumed
to be unable to transduce any Hh signal. Alternatively,
in smo mutant ovarioles, egg chamber budding
is sometimes arrested or defective, and
normal egg chambers completely enveloped by smo mutant
follicle cells have never been seen. These phenotypes might derive solely from an
insufficient supply of somatic cells, resulting directly from
impaired proliferation of smo mutant cells. However, the possibility cannot be dismissed that Hh signaling has a more
direct role in germline cyst encapsulation, promoting egg
chamber budding, or delaying somatic cell lineage decisions
until the appropriate developmental stage (Zhang, 2000).
Although Hedgehog proteins most commonly affect cell fate, they can also stimulate cell proliferation. In humans several distinctive cancers,
including basal-cell carcinoma, result from mutations that aberrantly activate Hh signal transduction. In Drosophila, Hh directly stimulates proliferation of
ovarian somatic cells. Hh acts specifically on stem cells in the Drosophila ovary. These cells cannot proliferate as stem cells in the absence
of Hh signaling, whereas excessive Hh signaling produces supernumerary stem cells. It is deduced that Hh is a stem-cell factor and it is suggested that human cancers due to excessive Hh signaling might result from aberrant expansion of stem cell pools (Zhang, 2001).
In adult Drosophila females, egg chambers are produced continuously in the germarium of each of the 15-18 ovarioles that are bundled together to form an ovary. In regions 1 and 2a of the germarium, 16-cell germline cysts develop from germline stem cells. Each cyst is enveloped by a monolayer of follicle cells in region 2b and separated from the next cyst by a short stalk as it buds from region 3 to form an egg chamber. Follicle and stalk cells derive from somatic stem cells that reside at the region 2a/2b border. When a somatic stem cell divides, one daughter retains a stem cell identity and continues to divide as a stem cell for several days. The other 'pre-follicle cell' daughter proliferates for about eight cycles as its progeny associate with germline cysts, pass posteriorly down the ovariole over a five to six day period, and differentiate into multiple specialized cell types. Hedgehog (Hh) is expressed selectively in specialized non-proliferating, somatic 'terminal filament' and 'cap' cells at the anterior tip of the germarium. Inactivation of Hh, using conditional hh alleles, arrests egg chamber budding, and causes germline cysts to accumulate in swollen germaria, suggesting that too few follicle cells are being produced. Conversely, excessive Hh signaling in germarial region 2 can be induced by temporally controlled activation of an hh transgene or by inactivation of the Hh receptor Patched (Ptc), and causes marked overproliferation of somatic cells, which accumulate between egg chambers (Zhang, 2001).
The proliferative response to excessive Hh signal transduction was investigated further by using antibodies against phospho-histone H3, which stains cells only during mitosis. High levels of Hh signal transduction were induced by inactivating patched (ptc) in marked somatic cell clones generated by heat-shock induced mitotic recombination. Ovaries were examined 8 d later to ensure that all proliferating somatic cells assayed derived from stem cells that were present at the time of clone induction. As each ovariole contains more than one somatic stem cell, this procedure generates some ovarioles containing only ptc mutant somatic cells and others that are mosaic for wild-type and ptc mutant cells. The number of mitotic somatic cells in germarial region 2 in ovarioles containing only ptc mutant somatic cells was twice that in wild-type ovarioles. A similar ratio was observed in region 3 of the germarium and in newly budded (stage 1-2) egg chambers, suggesting an early increase in proliferation of ptc mutant cells followed by wild-type rates of proliferation of a twofold enlarged cell population. Accordingly, the follicular monolayers surrounding stage 6 egg chambers in wild-type and ptc mutant ovarioles contained almost identical numbers of mitotic cells within numerically equivalent cell populations. In ovarioles mosaic for ptc mutant and wild-type somatic cells, the number of cells in mitosis was increased roughly 1.5-fold in region 2; again, this ratio was maintained in region 3 and in the earliest egg chambers. This is consistent with a cell-autonomous effect of ptc inactivation, affecting roughly half of the somatic cells in a mosaic germarium (Zhang, 2001).
To establish whether excessive Hh signaling accelerates somatic stem cell cycles or increases the number of stem cells in an ovariole, stem cells were counted by using mitotic recombination. Thus, in almost every ovariole examined, a loss of ptc activity led to a cell-autonomous doubling of somatic stem cell number. In 19 of these instances, the two ptc mutant stem cells were directly adjacent, suggesting that excessive Hh signal transduction allowed local expansion of a stem cell niche. In the remaining 12 cases the additional ptc mutant stem cell had migrated away from its presumed sister cell (Zhang, 2001).
Whether Hh is required for somatic stem cell maintenance or proliferation was investigated by using conditional hh alleles and by generating somatic cell clones lacking smoothened (smo) activity. Inactivation of smo universally blocks Hh signal transduction cell-autonomously. The results demonstrate that a cell that is unable to transduce an Hh signal cannot proliferate as a somatic stem cell. It is suspected that smo mutant somatic stem cells remain abnormally quiescent for up to 7-8 d and at some point during this period acquire the characteristics of a pre-follicle cell, proliferating normally in that capacity to produce a clone occupying roughly one-third of an egg chamber (Zhang, 2001).
The effects of Hh signaling on cell fate determination in Drosophila are mediated largely by altering the activity of the transcription factor Cubitus interruptus (Ci). The role of Ci in somatic stem cell proliferation was examined by inducing somatic clones lacking ci activity. As with smo, very few clones were recovered 8-10 d after clone induction and these clones occupied only a small proportion of the ovariole, indicating that stem cells cannot proliferate normally in the absence of ci activity. When the expression of a constitutively active derivative of Ci was induced by heat-shock-induced excision of a transcriptional terminator, ovarioles were recovered showing massive overproliferation of somatic cells, which accumulated between egg chambers as observed for ptc mutant ovarioles. Thus, the activity of Hh as a stem cell factor seems to depend on Ci-mediated regulation of transcription (Zhang, 2001).
The primitive gonad of the Drosophila embryo is formed from two cell types, the somatic gonad precursor cells (SGPs) and the germ cells, which originate at distant sites. To reach the SGPs the germ cells must undergo a complex series of cell movements. While there is evidence that attractive and repulsive signals guide germ cell migration through the embryo, the molecular identity of these instructive molecules has remained elusive. Evidence is presented suggesting that hedgehog (hh) may serve as such an attractive guidance cue. Misexpression of hh in the soma induces germ cells to migrate to inappropriate locations. Conversely, cell-autonomous components of the hh pathway appear to be required in the germline for proper germ cell migration (Deshpande, 2001).
Known cell-autonomous components of the Hh signaling pathway also appear to be required in germ cells for normal migration behavior. Germline clones were used to test four different hh pathway genes -- ptc, pka, smo, and fu. For all four, abnormalities in germ cell migration were observed in the progeny. In the case of both the ptc and smo germline clones, eggs fertilized by wild-type sperm developed into completely normal adults. Moreover, there are no apparent defects in the formation of the somatic gonad or in the pattern of Clift expression. These findings would support the view that the migration defects seen in ptcmat-zyg+ and smomat-zyg+ embryos arise from cell-autonomous deficiencies in the response to Hh by the germ cells. However, it should be pointed out that there could be some undetected nonautonomous problem in somatic hh signaling in these embryos that induces abnormalities in germ cell behavior (Deshpande, 2001).
As would be expected from the known properties of these four genes in other well characterized hh pathways, the phenotypes produced by ptc and pka germline clones are similar and quite distinct from those observed for smo and fu. Moreover, the migration defects observed in ptc/pka and smo/fu germline clones can be explained by the antagonistic role of these genes in the hh signaling pathway. In the absence of maternal ptc or pka, smo and its downstream effectors in the hh pathway are activated in the germ cells independent of the Hh ligand. As a consequence, many of the germ cells clump together as they begin passing through the midgut, and then remain in place instead of migrating toward the SGP cells. Additionally, the mitotic cycle in ptcmat- (and to a lesser extent pkamat-) germ cells is inappropriately activated. Up regulation of cell division has been observed in somatic tumors that lack ptc function and in ptc mutant C. elegans germ cells. In the case of smo and fu, the germ cells can't respond to the Hh ligand, and they are unable to detect or associate with the SGP cells, and instead migrate randomly through the mesoderm (Deshpande, 2001).
The function of the Dpp and Hh signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo has been analyzed. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). The similarities between vertebrate systems and Drosophila are discussed with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling (Chang, 2001).
Hh signaling is negatively regulated by Ptc, a membrane linked protein that, by binding to Hh ligand, becomes inactivated in cells receiving high levels of Hh. Ptc expression in the head resembles hh expression at an early stage. A wide antennal/pre-antennal stripe traverses the head in front of the cephalic furrow. During germband extension, this domain splits into two stripes. At the late extended germ band stage, ptc remains expressed in a large domain that corresponds to the anterior optic lobe (Chang, 2001).
Loss of hh results in a strong reduction of the head midline epidermis, a reduction in the size of the brain and optic lobe, and the total absence of the larval and adult eye primordium. Temperature-sensitive shift experiments of hhts2 embryos indicate that the phenocritical period for Hh function in Bolwig's organ development is between 4 and 7 hours. Aside from the larval eye, the primordium of the compound eye, which is marked from stage 12 onward by the expression of eyeless (ey), is also affected by the loss of hh. Heatshock induced overexpression of hh, as well as loss of ptc, causes an increase in larval eye neurons and optic lobe precursors. Interestingly, ectopic Hh activity is able to induce optic lobe and Bolwig's organ tissue in the head midline and thereby generate a cyclops phenotype similar to the condition described above for partial reduction of dpp. Applying heatshocks at different times of development indicates that the phenocritical period for the Hh induced cyclops is early, between 2.5 and 5 hours. Thus, heat pulses administered during this time cause fusion of the optic lobe and, at a lower frequency, of the larval eye without significantly increasing the number of optic lobe and larval eye cells. By contrast, later heat pulses (after 5 hours) lead to larval eye/optic lobe hyperplasia but no concomitant cyclops phenotype (Chang, 2001).
The finding that both loss of Hh and Dpp cause the absence of visual structures, and ectopic expression of Hh and partial loss of Dpp cause transformation of head midline epidermis into visual primordium, begs the question of how the two signaling pathways interact. In Drosophila compound eye development, hh expression is required to turn on dpp expression. To establish whether a regulatory relationship exists between Hh and Dpp signaling, the expression of dpp and pMAD was examined in the background of hh loss of function, as well as hh, ptc and Cubitus interruptus (Ci) expression in the background of dpp loss of function. Cells in which Dpp signaling is activated can be visualized by an antibody against phosphorylated MAD (pMAD) protein. Dpp RNA expression and pMAD are normal in a stage 5-9 hh-null background, indicating that Hh is not required to activate Dpp signaling in the embryonic head (Chang, 2001).
A model is proposed to explain the phenotypes resulting from manipulating Dpp, Hh and Ptc expression:
Patched regulates Drosophila head development by promoting cell proliferation in the eye-antennal disc. During head morphogenesis, Patched positively interacts with Smoothened, which leads to the activation of Activin type I receptor Baboon and stimulation of cell proliferation in the eye-antennal disc. Thus, loss of Ptc or Smoothened activity affects cell proliferation in the eye-antennal disc and results in adult head capsule defects. Similarly, reducing the dose of smoothened in a patched background enhances the head defects. Consistent with these results, gain-of-function Hedgehog interferes with the activation of Baboon by Patched and Smoothened, leading to a similar head capsule defect. Expression of an activated form of Baboon in the patched domain in a patched mutant background completely rescues the head defects. These results provide insight into head morphogenesis and reveal an unexpected non-canonical positive signaling pathway in which Patched and Smoothened function to promote cell proliferation as opposed to repressing it (Shyamala, 2002).
Thus, a novel pathway has been uncovered by which Ptc promotes proliferation of cells in the eye-antennal disc to generate the Drosophila head capsule. Ptc, together with the enigmatic transmembrane protein Smo, promotes activation of Babo, the Activin type I receptor, to stimulate cell proliferation. Previous studies have shown that Ptc is a repressor of Smo, and the interaction of Hh and Ptc relieves this repression on Smo, allowing Smo to activate downstream genes. Ptc signaling is also known to be a suppressor of cell proliferation and loss of function for Ptc in vertebrates, for example, leads to nevoid basal carcinomas. The results described here show that Ptc signaling, in concert with Smo, can also promote cell proliferation and that this is via activation of downstream genes. Thus, these results reveal an intriguing and non-canonical mode of action by this pathway during head morphogenesis (Shyamala, 2002).
The loss of the head capsule in ptc mutants is not due to cell death, no inappropriate and massive cell death has been observed in the eye-antennal disc by the TUNEL assays. However, a lack of BrdU incorporation is observed as well as fewer phospho-histone-positive cells in the eye-antennal disc. Lack of differentiation of cells of the eye-antennal discs can also give rise to similar head capsule defects. For example, pharate adults mutant for the headcase gene show severe head capsule defects with resemblance to ptc mutants. However, in headcase mutants, the morphology, the size and the shape of the eye-antennal discs are normal and the head capsule defects appear to be due to a failure in the differentiation of cells of the eye-antennal disc. In ptc mutants, the morphology, organization, and size of the eye-antennal disc are severely affected by late 3rd instar larvae and the primary cause for the head capsule defects is loss of cell proliferation. This conclusion is further supported by the fact that an activated form of Babo completely rescues the head capsule defects in ptc mutants. babo is a known player in promoting cell proliferation and is required only for cell proliferation but not for cell differentiation in the imaginal discs. Moreover, in vitro culture of eye-antennal discs indicate that the differentiation per se is not affected in ptc mutants. Therefore, it is concluded that Ptc promotes cell proliferation in the eye-antennal disc during head development (Shyamala, 2002).
Previous studies indicate that Ptc is likely to complex with Smo and repress Smo from activating downstream target genes. Binding of Hh to Ptc frees Smo from Ptc repression, which then goes on to activate downstream target genes. Thus, Ptc has been always viewed as a suppressor of gene activity via suppressing Smo. For example, during the development of the embryonic nerve cord, loss of ptc activity leads to missing RP2 neurons. This is due to the ectopic activation of Gsb in the neuroectoderm from which the RP2 precursor neuroblast (NB4-2, a row 4 NB) delaminates; ectopic Gsb prevents Wingless signaling from specifying NB4-2 identity and therefore the loss of RP2 neurons. Consistent with the possibility that Smo is downstream of Ptc, ectopic expression of Gsb in row 4 in ptc mutants and the consequent loss of RP2 neurons is rescued in ptc, smo double mutants. If this signaling also occurs during the head development, loss of Ptc will lead to inappropriate activation of Smo, leading to the head capsule defects; loss of Smo activity in a ptc mutant background, therefore, should suppress the head capsule defects. However, reducing the dose of Smo in a ptc mutant background (smo/+, ptcgal4/ptcnull), instead of suppressing the head defects (or at least reducing the severity), enhances the head capsule defects. Moreover, loss of Smo activity leads to the same head capsule defects as in ptc mutants (Shyamala, 2002).
Previous results have indicated that Ptc might negatively regulate levels of Smo via vesicular trafficking of Smo from the cell surface. Thus, in ptc mutants it has been inferred that the level of Smo on the membrane is high, leading to the inappropriate activation of downstream target genes. That a similar mechanism might operate during head capsule development is unlikely for the following reasons: (1) reducing the dose of smo in ptc mutant background enhances the phenotype; (2) in one of the ptc alleles, ptcS2, the mutation is an amino acid change from charged to neutral in the sterol-sensing domain. ptcS2 fully complements ptchdl and the transheterozygotes have no head capsule defects. Moreover, in ptchdl/ptcnull mutant eye-antennal disc, the level of Smo is not upregulated. Based on these results, it is concluded that a positive signaling by Ptc and Smo regulates cell proliferation during head development (Shyamala, 2002).
In the conventional Ptc-signaling, interaction of Hh with Ptc relieves the repression on Smo, thus allowing Smo to function. When Hh is ectopically expressed, it interacts with Ptc to relieve the repression on Smo. This in turn is thought to cause phenotypes in hh gain-of-function situations. Thus, in the CNS, for example, loss of Ptc activity from the RP2 neuronal precursor cell leads to missing RP2 neurons; ectopic expression of Hh in adjacent rows of cells leads to loss of RP2 neuron via inappropriate activation of Gsb in the neuroectoderm from which NB4-2 is delaminated. The results described in this paper, that during head development gain-of-function Hh mimics a loss of function ptc phenotype, are not inconsistent with the finding that Ptc, together with Smo, promotes cell proliferation. That is, ectopic expression of Hh will bind to Ptc and this will interfere with the positive signaling by Ptc and Smo. One possibility is that Ptc and Smo are physically associated with one another, and binding of Hh to Ptc will break this physical association, rending Ptc or Smo unable to positively regulate cell proliferation in the eye-antennal disc (Shyamala, 2002).
The results indicate that Ptc-Smo signaling leads to the activation of Babo. During Activin signaling, Activin binds to Activin type II receptor, which promotes physical interaction between type II and type I receptors and the phosphorylation of type I receptor. Both type I and type II receptors are transmembrane serine/threonine kinases. Phosphorylation of the type I receptor results in the activation of its kinase activity and the phosphorylation of downstream transcription activators such as the Smad proteins, resulting in their nuclear localization. In Drosophila, analysis of null mutants for the type I receptor babo, as well as analysis of babo germline clones, indicates that babo is not required during embryogenesis but is essential during pupal development and adult viability. The major defect in babo mutants is a reduction of cell proliferation in the imaginal discs and brain tissue. It has also been shown that in tissue culture experiments, a constitutively active form of Babo can signal to vertebrate TGF-ß/Activin, but not to BMP-responsive promoters. The activated Babo then interacts with Drosophila Smad2 to effect the nuclear localization of this transcription factor (Shyamala, 2002).
These results, that expression of an activated form of Babo in the ptc-expression domain in the eye-antennal disc of ptc mutants completely rescues the head capsule defects, indicates that Ptc-Smo signaling ultimately leads to activation of Babo and promotes cell proliferation in the eye-antennal disc. Since babo and ptc show transheterozygous interaction, it is tempting to speculate that the interaction between Ptc and Babo might be direct. A transheterozygous interaction is generally observed in several cases where the two proteins associate with one another, in cases such as the receptor-ligand pairs Notch and Delta. However, it is also possible that Ptc-Smo signaling and Babo signaling represent parallel pathways that converge at the point of cell cycle control. In this scenario, partial reduction in each could have a synergistic negative affect on cell proliferation, while overexpression of one (i.e. activated Babo) could compensate for loss of the other. Yet another possibility would be that the Pt-Smo pathway activates one of the Activin-like ligands. While the results indicate that there is no transheterozygous genetic interaction between ptc and punt (the inferred type II receptor for Activin), the possibility cannot be ruled out that the Ptc-Smo pathway does not interact with Punt. This is due to the fact that a lack of transheterozygous interaction does not mean that the two players do not interact, as it actually depends on what is limiting. Nonetheless, the finding that Ptc, together with Smo stimulates cell proliferation and the interfacing of Ptc-signaling with Babo-signaling in this process provides new insight into the process of head development (Shyamala, 2002).
Characterization of different alleles of the Hedgehog receptor patched (ptc) indicates that they can be grouped into several classes. Most mutations result in complete loss of Ptc function. However, missense mutations located within the putative sterol-sensing domain (SSD) or C terminus of ptc encode antimorphic proteins that are unable to repress Smo activity and inhibit wild-type Ptc from doing so, but retain the ability to bind and sequester Hh. Analysis of the eye and head phenotypes of Drosophila in various ptc/ptctuf1 heteroallelic combinations shows that these two classes of ptc alleles can be easily distinguished by their eye phenotype, but not by their head phenotype. Adult eye size is inversely correlated with head vertex size, suggesting an alteration of cell fate within the eye-antennal disc. A balance between excess cell division and cell death in the mutant eye discs may also contribute to final eye size. In addition, contrary to results reported recently, the role of Hh signaling in the Drosophila head vertex appears to be primarily in patterning rather than in proliferation, with Ptc and Smo having opposing effects on formation of medial structures (Thomas, 2003).
Antimorphic alleles of ptc were initially identified by the severity of their wing phenotype when heteroallelic with ptctuf1. Such trans-heterozygotes exhibit large outgrowths of the anterior wing, consistent with activation of dpp due to ectopic Hh signaling, whereas in the hemizygous condition ptctuf1 shows only a mild anterior outgrowth. By contrast, the eye phenotype appears, at least at first sight, to be much more severe in LF/ptctuf1 mutants than in AN/ptctuf1 mutants. However, closer analysis reveals that the two classes of alleles have distinct effects, the eyes of AN/ptctuf1 adults being significantly larger than those of wild type, rather than simply less reduced than those of LF/ptctuf1 mutants. However, the head defects typical of AN/ptctuf1 trans-heterozygotes do appear to be mild versions of those seen in LF/ptctuf1 mutants. Because antimorphic Ptc proteins are distinguished by their ability to sequester the Hh ligand, this implies that excessive Hh diffusion, rather than ectopic pathway activation due to Smo derepression, is the principal cause of the head phenotypes such as ectopic ocelli. In summary, it appears that the difference between allele class phenotypes in wing, eye, and head reflects the different relative impact of cell-autonomous ectopic pathway activation vs. excess Hh diffusion in the three different structures (Thomas, 2003).
In the canonical Hh signaling pathway, Ptc functions as a negative regulator both by sequestering Hh ligand and by inhibiting Smo activity. By contrast, evidence has been presented suggesting that Ptc and Smo act in concert to promote growth in the head, a function that is opposed by the activity of Hh. Reexamination of this issue does not support such an atypical interaction between Ptc and Smo in the head; moreover, it suggests that the predominant role of Hh signaling in the head is in patterning rather than in proliferation (Thomas, 2003).
The eye disc contains two domains of apoptosis: one immediately anterior to the MF that is regulated by Eya and one posterior to the MF whose function is unknown. The latter domain of cell death is promoted by Hh signaling from photoreceptors. The milder increase in cell death seen in AN/ptctuf1 trans-heterozygotes perhaps reflects the efficient sequestration of Hh in such mutants, suggesting that the range of Hh diffusion may be important in influencing the proportion of cells that die behind the MF. Normally, a large amount of cell death, necessary for the elimination of two to three excess pigment cells per ommatidium, occurs in pupal eye discs. Cell death behind the MF may have a similar function, perhaps in removing cells mis-specified during differentiation. It is possible that Hh could regulate apoptosis through activation of a molecule at short range behind the MF. Alternatively, cell death may not depend directly upon Hh, but rather upon the presence of increased numbers of mis-specified cells in ptc mutants that may result in cell death occurring to regulate differentiation effectively (Thomas, 2003).
The earliest known role of Hh in the eye imaginal disc is to help specify the dorso-ventral organizer. Incorrect DV specification compromises the function of N in promoting growth, resulting in small eyes. However, the small-eye phenotype seen in LF/ptctuf1 appears to be unconnected to this process. Although some disorganization of the equator is seen in ptc trans-heterozygotes, it is not significant, and its presence in both classes of mutant indicates that the effect, if any, on eye size is small (Thomas, 2003).
The two major targets of Hh signaling during MF progression are dpp and ato. The data indicate that although dpp is ectopically activated in ptc trans-heterozygotes, ato is not. This is unexpected, since Hh signaling activates the initial expression of ato, so an increase might be anticipated to expand the ato expression domain into more anterior regions, while maintaining or even increasing the level of expression. Conversely, reducing the activity of ptc in the hh1 mutant rescues the expression of ato, but not that of dpp. There are several possible explanations for these findings. (1) ato is an indirect target of Hh signaling and the mediators of Hh activity in this context are unclear. It is likely that other factors, in addition to those directly induced by Hh, are necessary for ato expression, any one of which may be limiting. (2) dpp may respond to lower levels of Hh pathway activation more than genes upstream of ato. In the wing disc, dpp is activated by relatively low levels of Hh anterior to the AP border, whereas other Hh target genes such as collier require a higher level of pathway activation. In ptc trans-heterozygotes some Ptc activity is retained and hence the very highest level of Hh signaling cannot be reached. (3) Dpp in its role as an inducer of the preproneural state can actually inhibit the expression of ato through activation of the proneural repressors h and emc. In ptc trans-heterozygous discs, the domain of h expression does appear to be expanded, suggesting a possible explanation for the observed downregulation of ato expression. A fourth possibility that has not been tested is that the increased level of Hh signaling in ptc trans-heterozygote discs results in an expansion of the domain of rough (ro) expression. Ro is induced by high-level Hh signaling at the posterior margin of the MF, but, if expressed at excessive levels (as in the roDom mutant), causes a downregulation of ato expression. Although a severe reduction in ato expression such as that caused by roDom can result in furrow arrest, the significance of a mild downregulation of expression is unknown (Thomas, 2003).
The two classes of mutants both show an increase in dpplacZ expression relative to wild type. However, the domain of ectopic expression differs significantly between allele types, suggesting a difference in the way in which the pathway is activated in the two classes of mutant. Because LF ptc alleles cannot sequester Hh efficiently, the broad band of ectopic dpplacZ seen ahead of the MF may be caused by excessive diffusion of Hh anteriorly. In contrast, the AN/ptctuf1 trans-heterozygotes can sequester Hh efficiently and consequently demonstrate only phenotypes caused by autonomous ectopic pathway activation (Thomas, 2003).
Despite the rescue of adult-eye phenotype observed in ptc/ptctuf1;hh1 double mutants, the expression of dpplacZ was not restored in the center of the disc. Since Dpp does not play a major role in MF progression, the lack of expression in this situation may not have a significant effect. Alternatively, excessive dpp expression at the margins may allow the protein to diffuse medially into the disc, thus aiding MF progression in an unconventional way (Thomas, 2003).
Dpp is known to have several functions in the eye disc, all of which, when modified, can influence the final size and shape of the adult eye. However, despite the disparity between the patterns of dpplacZ expression in the two types of trans-heterozygote, surprisingly little difference is detected downstream of Dpp. Although ectopic dpp expression has been shown to induce ectopic MFs, this does not occur in the ptc trans-heterozygotes, presumably because the ectopic Dpp is either not high enough or not expressed in the right place (Thomas, 2003).
In addition to its effect on furrow initiation, Dpp is also responsible for defining the eye field via inhibition of Wg and for inducing cell cycle arrest ahead of the furrow. Small-eye mutants do show an increased head vertex size, suggesting that an eye-to-vertex fate change has occurred. However, ptc trans-heterozygotes do not display critical differences either from wild type or between allele classes in the distribution of Wg in second instar eye discs. In addition, dpp expression is actually expanded in eye discs of small-eye mutants, indicating that processes other than Wg/Dpp antagonism must be involved in specification of eye vs. head domains (Thomas, 2003).
Ectopic Dpp ahead of the furrow does not appear to induce premature cell cycle arrest and therefore cannot explain the reduced-eye phenotypes observed in LF/ptctuf trans-heterozygotes. However, when compared to wild type, ptc mutants do show an increase in cell divisions ahead of the furrow. It is suggested that in addition to an eye/head vertex specification defect, LF/ptctuf1 trans-heterozygotes may exhibit a small-eye phenotype due to excessive cell death, despite some increase in cell divisions ahead of the furrow. Conversely, in DN/ptctuf1 trans-heterozygotes, increased proliferation could overcompensate for increased cell death, leading to larger eyes. This suggests that the adult-eye phenotype is at least partially dependent upon a balance between cell division and cell death in the disc, in addition to an eye-to-head fate change (Thomas, 2003).
patched:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions
| Developmental Biology
| References
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