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 formation of segmental grooves during mid embryogenesis in the Drosophila epidermis depends on the specification of a single row of groove cells posteriorly adjacent to cells that express the Hedgehog signal. However, the mechanism of groove formation and the role of the parasegmental organizer, which consists of adjacent rows of hedgehog- and wingless-expressing cells, are not well understood. This study reports that although groove cells originate from a population of Odd skipped-expressing cells, this pair-rule transcription factor is not required for their specification. It was further found that Hedgehog is sufficient to specify groove fate in cells of different origin as late as stage 10, suggesting that Hedgehog induces groove cell fate rather than maintaining a pre-established state. Wingless activity is continuously required in the posterior part of parasegments to antagonize segmental groove formation. These data support an instructive role for the Wingless/Hedgehog organizer in cellular patterning (Mulinari, 2009).
It has been reported that segmental groove formation requires the activity of engrailed (en) and hh and that en has a function that is independent of its role in hh activation. More recently, it was been found that en is not expressed in groove cells, thus creating a non-cell-autonomous requirement for en. To address this issue, the role of hh and en in segmental groove formation was reinvestigated (Mulinari, 2009).
It was found that segmental grooves do not form in hh mutants. When hh was overexpressed, the four to five cell rows posterior to the Hh source constricted apically, elongated their apical-basal axis and took on a shape characteristic of segmental groove cells. Very similar cell behavior was observed in patched (ptc) mutants or when activated Ci, which mediates hh activity, was expressed. These observations suggest that Hh can organize segmental groove formation. No cell constrictions were observed in the ventral epidermis, indicating that a different mechanism might regulate cell shape there (Mulinari, 2009).
To address the proposed hh-independent function of en, en, invected (inv) double mutants were investigated in which hh expression was maintained using prd-Gal4. Segmental grooves were rescued in these mutants, suggesting that en is not required for segmental groove formation independent of its role in hh activation. By contrast, it was found that en represses groove cell behavior when ectopically expressed together with hh. A previous study that reported a requirement of en in groove formation was based on the analysis of en, inv, wg triple mutants, in which hh expression was maintained but did not rescue groove formation. This result was confirmed, but it is proposed that wg may be required in en mutants to allow the morphological differentiation of grooves (Mulinari, 2009).
Analysis of ptc mutants, or embryos overexpressing hh, reveals that a broad region of cells posterior to the en expression domain are specified as groove cells. However, groove-like invaginations form only at the edges of these regions. This is even more obvious in double mutants of ptc and the segment polarity gene sloppy paired 1 (slp1), which is required for maintained wg expression. In slp1, ptc mutants, wg expression fades prematurely and Hh signaling is constitutively active. This results in a substantial expansion of the number of groove cells. However, furrows differentiate only at the edges of groove cell populations. It is proposed that the morphological differentiation of segmental grooves can occur only at the interface between groove and non-groove cells (Mulinari, 2009).
To test this, wg, ptc double mutants were used in which Hh signaling is active throughout the epidermis and all cells take on a groove fate. Interestingly, these embryos did not differentiate grooves. A similar observation has been reported in en, inv, wg mutants, in which hh expression is sustained, leading to the suggestion that en might be required for groove specification (Mulinari, 2009).
Analysis of cell behavior in wg, ptc mutants showed, however, that cells throughout the tissue constrict their apices but fail to form invaginating furrows. The failure of wg, ptc mutants and en, inv, wg; UAS-hh embryos to differentiate grooves might be due to the absence of non-groove cells in the epidermis and the concomitant absence of an interface with groove cells (Mulinari, 2009).
The pair-rule gene odd is initially expressed in 4- to 5-cell wide stripes in even-numbered parasegments. At early gastrulation, odd expression expands to segmental periodicity and is subsequently refined to a single row of prospective groove cells located posterior to en. Continued expression of odd in these cells requires hh. In odd5 mutant embryos, grooves are unaffected in odd-numbered parasegments, but partially missing in even-numbered parasegments, and residual grooves coincide with regions in which odd expression is detectable (Vincent, 2008). These observations have been interpreted as indicating that groove fate might be specified prior to the requirement of Hh and differentiation of the groove. Thus, the later activity of Hh might not induce, but merely maintain, groove cell identity that has been pre-established in the odd-expressing cell population (Vincent, 2008). However, this hypothesis is based on the presumption that odd has a function in groove cell specification and this has not been demonstrated (Mulinari, 2009).
Residual grooves in odd5 mutants have been attributed to the hypomorphic nature of the odd5 allele; however, the molecular lesion in odd5 is unknown. Therefore the nucleotide sequence of odd5 was determined and a substitution was found that mutates codon 84 from CAG to a TAG stop codon. The resulting truncated peptide, which lacks all four putative zinc fingers encoded by wild-type odd, is no longer restricted to the nucleus but uniformly distributed in the cell. Thus, odd5 is likely to be a null allele (Mulinari, 2009).
To exclude the possibility that groove formation may be rescued by read-through of the stop codon in odd5 mutants, or that odd may be required redundantly, segmental grooves were investigated in Df(2L)drmP2 mutants, in which odd and its sister genes drumstick (drm) and sister of odd and bowl (sob) are entirely deleted. In these embryos, normal grooves formed in odd-numbered parasegments in the complete absence of odd function (Mulinari, 2009).
Next even-numbered parasegments were investigated in which grooves are partially missing. odd encodes a transcriptional repressor that regulates the expression of other segmentation genes in the early embryo. In odd mutants, derepression of the en activator fushi-tarazu in even-numbered parasegments results in the formation of an ectopic en stripe posterior to the normal stripe. Simultaneously, wg expands anteriorly and becomes expressed adjacent to the ectopic en-expressing cells. This results in the formation of an ectopic parasegment boundary with reversed polarity. Thus, the outward-facing edges of both en stripes are genetically anterior and lined by wg-expressing cells that do not form grooves. The inward-facing edges of the normal and ectopic en stripes fuse in some areas, and these corresponded to areas in which grooves were missing, as cells that were genetically posterior to en and could respond to the Hh signal had been replaced by en-expressing cells. The fusion of normal and ectopic en stripes was more severe in Df(2L)drmP2 mutants; however, islands of invaginating groove cells could still be observed, demonstrating that groove fate is specified in the absence of odd, drm and sob function in all parasegments. It is concluded that all cells that are genetically posterior to en are specified as groove cells in the absence of odd function and the partial absence of grooves in even-numbered parasegments in odd mutants is a secondary consequence of the pair-rule phenotype of these embryos. The slightly more severe pair-rule phenotype seen in Df(2L)drmP2 mutants might be due to a contribution from one of the odd sister genes, most likely sob, to pair-rule function, or could be caused by low-level read-through of the stop codon in the odd5 allele (Mulinari, 2009).
Finally, to investigate whether odd is sufficient to trigger cell shape changes, a UAS-odd transgene was expressed either alone or together with hh in the epidermis. No induction of groove cell behavior other than that triggered by hh was observed. Together, the data show that odd plays no essential role in groove cell specification and that odd paralogs are unlikely to act redundantly in this process (Mulinari, 2009).
The identification of odd as a groove cell marker led Vincent to suggest that groove fate might be specified prior to Hh requirement and that Hh may merely maintain groove fate instead of having an inducing role (Vincent, 2008). This study demonstrate that grooves are specified in the absence of odd function; however, this could be due to an odd-independent, early-acting mechanism present in the cells from which grooves arise (Mulinari, 2009).
In order to address whether groove fate is pre-established in the odd-expressing cell population, it was asked if groove fate could be induced in cells of a different origin at a later point in time. lines (lin) mutants were used in which late wg expression is altered resulting in the formation of an ectopic segment boundary at the anterior edge of the en domain in the dorsal epidermis. Importantly, the early expression of pair-rule or segment polarity genes is not affected (Mulinari, 2009).
In lin mutants, ectopic expression of the groove marker odd was initiated at stage 12 in a single row of groove-forming cells anterior to en that are derived from a previously non-odd-expressing cell population that does not contribute to grooves in the wild type. Ectopic grooves require hh as they were not induced in hh, lin double mutants, and ectopic odd expression was not induced in this background. An increase in hh levels in lin mutants resulted in the specification of groove fate in all cells except those expressing en. These results suggest that hh is sufficient, late in development, to specify groove cell fate in cell populations of different origins and that earlier-acting factors present in the population of odd-expressing cells posterior to en are not required. Very similar results have been reported by Piepenburg (2000), who showed that segment border cells form solely in response to the Hh signal that emanates from the en domain (Mulinari, 2009).
The findings are consistent with the role of Hh in the regulation of cell shape in other systems. Thus, during Drosophila eye development, Hh has been shown to control cell shape in the morphogenetic furrow, and Hh activation in other tissues is sufficient to induce apical constriction and groove formation. It is likely that Hh plays a similar role in tissue morphogenesis in other organisms. During neural tube closure in vertebrates, cells undergo similar shape changes involving apical-basal elongation and apical constriction, which is likely to be in response to Hh sources in the notochord and floor plate. Accordingly, knockout of sonic hedgehog is associated with defects in neural tube closure in mice. These observations suggest that Hh might be a principal inducer of cell shape across species (Mulinari, 2009).
It has previously been established that wg antagonizes the activity of hh in the specification of segment border cells (Piepenburg, 2000). However, it is not clear whether wg has a similar role in segmental groove formation, and a late requirement of wg to antagonize Hh-mediated groove specification has been questioned (Vincent, 2008). To investigate a direct role of wg in groove specification, a dominant-negative form of the transcription factor pan (panDN), which suppresses Wg signaling, was expressed. For this, pnr-Gal4, which initiates expression in the dorsal epidermis at stage 10-11 and thus does not affect early wg function, was used. Embryos that express panDN formed a single row of ectopic groove cells anterior to the en domain, confirming the results in lin mutants. Strikingly, inactivating Wg signaling and increasing Hh levels at the same time by co-expression of panDN and hh resulted in the expansion of groove fate to all cells except those expressing en. These results show that Wg signaling is required after stage 10 to repress groove specification anterior to en, thus making the activity of Hh asymmetric. These results also confirm observations that Hh is sufficient to induce groove fate in cells from different positions along the anterior-posterior axis and suggest that groove fate is not determined before stage 10 (Mulinari, 2009).
To confirm the ability of wg to repress groove fate, wg was expressed posterior to en in cells that normally take on groove fate. This resulted in the loss of Odd from many cells, suggesting that wg indeed antagonizes hh activity. Interestingly, these cells still formed grooves. However, these grooves appeared much earlier than segmental grooves, suggesting that they are ectopic parasegmental grooves caused by ectopic wg expression, as recently suggested (Larsen, 2008). Together, these data therefore support the contention that Wg signaling is required to repress Hh-mediated induction of groove fate after stage 10, thus permitting the formation of segmental grooves posterior, but not anterior, to en in the wild type (Mulinari, 2009).
patched:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions
| Developmental Biology
| References
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.