rhomboid
Rhomboid is initially expressed before the completion of cellularization in lateral stripes
within the presumptive neuroectoderm. The stripes are seven to eight cells wide, with irregular boundaries dorsally and ventrally. A dorsal stripe appears shortly and the ventral stripes become sharply delimited. There are two stripes in the head, perpendicular to the long axis in the embryo. The two lateral stripes meet at the ventral midline during gastrulation. There is later expression in a limited number of chordotonal neuron precursors, as well as expression in the tracheal pits. At the completion of germ-band retraction rhomboid is expressed in the ectoderm at the anterior border of each segment.
Additional expression is seen in the proventriculus (where esophagus and midgut meet), in the hindgut, and at the base of posterior spiracles (Bier, 1990).
The dorsal median cells are unique mesodermal cells that reside on the surface of the ventral nerve
cord in the Drosophila embryo. The Buttonless homeodomain protein is specifically expressed in these
cells and is required for their differentiation. Proper buttonless gene
expression and dorsal median cell differentiation require signals from underlying CNS midline cells.
Thus, dorsal median cells fail to form in single-minded mutants and do not persist in slit mutants.
Through analysis of rhomboid mutants and targeted rhomboid expression, it has been shown that the EGF
signaling pathway regulates the number of dorsal median cells. wingless-patched double
mutants exhibit defects in the restriction of dorsal median cells to segment boundaries and alterations in
CNS midline cell fates (Zhou, 1997).
The formation of the tracheal network in Drosophila is driven by stereotyped migration of cells from
the tracheal pits. No cell divisions take place during tracheal migration and the number of cells in each
branch is fixed. This work examines the basis for the determination of tracheal branch fates, prior to
the onset of migration. The EGF receptor pathway is activated by localized processing of
the ligand Spitz in the tracheal placodes and is responsible for the capacity to form the dorsal trunk
and visceral branch. Prominent double phosphorylation of Erk (Rolled) is detected in the tracheal placodes at stage 10-11. This pattern is Efgr-dependent and is abolished in rhomboid mutants. The double phosphorylated Erk domain is broader than the region of rhomboid expression. Since Rhomboid is known to regulate Spitz processing, this pattern probably reflects the diffusion of the secreted form of Spitz originating within the rhomboid-expressing cells, in the central part of the placode. In mutants for Egfr, tracheal pits appear normal, although certain tracheal branches fail to develop: specifically, the dorsal trunk and visceral branch are missing or incomplete. spalt mutants show specific defects in the migration of dorsal trunk cells, pointing to an important role for spalt in subdivision of tracheal fates. The Dpp pathway is induced in the tracheal pit by local
presentation of Dpp from the adjacent dorsal and ventral ectodermal cells. This pathway patterns the
dorsal and lateral branches. Elimination of both Dpp and Egfr pathways blocks migration of all tracheal branches.
Antagonistic interactions between the two pathways are demonstrated. The opposing activities of two
pathways may refine the final determination of tracheal branch fates. Egfr-dependent activation of Erk (Rolled) in the tracheal placode precedes the activation of the same pathway by Breathless. Only after Egfr induction is diminished, does a new double phosphorylated Erk pattern appear, induced by Breathless. It is proposed that two opposing gradients of Dpp and Spitz are operating within the placode. the cells in the center of the tracheal placode encounter high concentrations of secreted Spitz, and low or negligable levels of Dpp. Conversely, the cells located at the dorsal and ventral domains of the placode encounter high concentrations of Dpp and low levels of secreted Spitz. Therefore, induction by the Egfr and Dpp pathways creates three subsets of cells: dorsal, central and ventral (Wappner, 1997).
In addition to prominent CNS midline expression, the single minded gene is also expressed in clusters of mesodermal cells that arise adjacent to the midline, migrate laterally during germ band retraction and contribute to ventral muscle fibers. The EGF signaling pathway regulates this set of mesodermal cells that expresses rhomboid. Mesodermal expression of rho is transient and eliminated well before muscle fiber formation, suggesting that at least some of the muscle defects in rho mutants may result from earlier patterning defects in specific mesodermal precursor cells. While the function of sim in developing is unclear, it is of interest that the mesodermal sim expression is conserved in vertebrates. Thus, the mouse Sim-1 gene is prominently expressed in paraxial mesodermal cells of the developing lateral somitic compartment. Taken together, these data define a novel neuroectoderm to mesoderm signaling pathway and suggest that unique mesodermal cell types are specified by a combination of
midline and segmental cues (Zhou, 1997).
Muscle development initiates in the Drosophila embryo with the segregation of single progenitor cells, from each of which a complete set of myofibers arises. Each progenitor is assigned a unique fate, characterized by the expression of particular gene identities. The Drosophila Epidermal growth factor receptor (Egfr) provides an inductive signal for the specification of a large subset of muscle progenitors. In the absence of the receptor or its ligand, Spitz, specific progenitors fail to segregate. The resulting unspecified mesodermal cells undergo programmed cell death. In contrast, receptor hyperactivation generates supernumerary progenitors, as well as the duplication of at least one Spitz-dependent myofiber. The requirement for Egfr occurs early in muscle cell specification, as early as five to seven hours after fertilization. The development of individual muscles is differentially sensitive to variations in the level of signaling by the Epidermal growth factor receptor. Such graded myogenic effects can be influenced by alterations in the functions of Star and Rhomboid. In addition, muscle patterning is dependent on the generation of a spatially restricted, activating Spitz signal, a process that may rely on the localized mesodermal expression of Rhomboid. Thus, Epidermal growth factor receptor contributes both to muscle progenitor specification and to the diversification of muscle identities (Buff, 1998).
Star, which is known to interact with Egfr, modifies myogenic signaling by Egfr. Ectopic mesodermal expression of DNDER yields a sensitized background in which to quantitate genetic interactions with Star. One copy of UAS-DNDER caused a partial reduction in the development of DA1 and VA2. This effect is suppressed by co-expression of full-length Egfr or Star. Ectopic expression of Star or full-length Egfr in a wild-type genetic background had no effect on muscle development. The UAS-Star results also indicate that Star is required autonomously for Egfr function in the mesoderm. Star dominantly enhances the effect of DNDER on muscles DA1 and VA2, suggesting that Star is normally limiting for muscle development. Rhomboid is also required for muscle DA1 formation and is expressed in the mesoderm in proximity to the DA1 progenitor. As was found for spi, Star and Egfr, rho is also required for development of the Eve-expressing muscle DA1 precursor but not for formation of the adjacent pericardial cells. Because Rho is a positive regulator of Egfr and its expression is frequently localized to sites where Egfr signaling is active, the expression of rho in the vicinity of DA1 was examined during the course of its development. rho transcripts are found in segmentally repeated dorsal mesodermal cells in stage-11 embryos. These cells are located at the peaks of the mesodermal crests that lie between the tracheal pits, precisely where the Eve-expressing P2 and P15 progenitors and their founders arise. By double-labeling with Rho and Eve antibodies, it was found that Rho is co-expressed with Eve in P2. This is a particularly intriguing finding since the specification of P2 (the pericardial progenitor) precedes that of P15 (the muscle DA1 progenitor): these two cells segregate in very close proximity to each other, and only P15 is Egfr-dependent. Even under conditions where muscle DA1 forms in the absence of Eve pericardial cells, such as with partial inhibition of Heartless activity, Rho is expressed in a mesodermal cell that resembles a normal P2 but lacks Eve. Given the known effects of Rho in modifying Egfr activity in other developmental contexts, the temporal and spatial expression of Rho in the dorsal mesoderm is consistent with a functional role for Rho in the Egfr signaling responsible for P15 induction (Buff, 1998).
The transcription factors
encoding genes tailless (tll), atonal (ato), sine oculis (so),
eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in
establishing the Drosophila embryonic visual system. The
embryonic visual system consists of the optic lobe
primordium, which, during later larval life, develops into
the prominent optic lobe neuropiles, and the larval
photoreceptor (Bolwig's organ). Both structures derive
from a neurectodermal placode in the embryonic head.
Expression of tll is normally confined to the optic lobe
primordium, whereas ato appears in a subset of Bolwigs
organ cells that are called Bolwigs organ founders.
Phenotypic analysis of tll loss- and gain-of-function
mutant embryos using specific markers for Bolwigs
organ and the optic lobe, reveals that tll functions to drive cells to an
optic lobe fate, as opposed to a Bolwigs organ fate. Similar
experiments indicate that ato has the opposite effect,
namely driving cells to a Bolwigs organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells
to respond to signaling arising from ato-expressing
Bolwigs organ pioneers. The data further suggest that the
Bolwigs organ founder cells produce Spitz (the Drosophila
TGFalpha homolog) signal, which is passed to the neighboring
secondary Bolwigs organ cells where it activates the Epidermal growth factor receptor
signaling cascade and maintains the fate of these secondary
cells. The regulators of tll expression in the embryonic
visual system remain elusive, no
evidence for regulation by the 'early eye genes' so, eya and
ey, or by Egfr signaling is found (Daniel, 1999).
The Drosophila visual system comprises the adult compound eye,
the larval eye (Bolwigs organ) and the optic lobe (a part of the brain). All of these
components are recognizable as separate primordia during late
stages of embryonic development. These components originate from a small,
contiguous region in the dorsal head ectoderm. During the
extended germband stage, the individual components of the
visual system can be distinguished morphologically as well as by
spatially localized expression of the homeobox gene so and the adhesion molecule Fas II. Initially centered as an unpaired, oval domain
straddling the dorsal midline, the anlage of the visual
system subsequently elongates in the transverse axis and narrows
in the anteroposterior axis. By late gastrulation (stage 8), the
anlage occupies two bilaterally symmetric stripes that are anterior and
adjacent to the cephalic furrow. The domain of so
expression at this stage contains two regions with a high
expression level [olex (the external fold of the optic lobe) and olin]. Only these two regions
will ultimately give rise to the optic lobe and Bolwigs organ; the
so-positive cells dorsal and posterior to these domains will either form
part of the dorsal posterior head epidermis (dph) or undergo apoptotic cell death.
During the extended germband stage, the
anlage of the visual system expands further
ventrally until, around stage 10, it reaches the
equator (50% in the dorsoventral axis) of the
embryo. Shortly thereafter, olin, the
portion of the anlage that will give rise to most of
the optic lobe and Bolwigs organ, reorganizes
into a placode of high cylindrical epithelial cells
that differ in shape from the surrounding more
squamous cells of the head ectoderm.
During stage 12, this placode starts to invaginate,
forming a V-shaped structure with an anterior lip
(olal) and a posterior lip (olpl). Bolwigs organ,
which consists of a small cluster of sensory
neurons, derives from the basal part of the
posterior lip and can be recognized during stage
12 as a distinct, dome-shaped protrusion. During stage 13,
invagination of the optic lobe separates it from the
head ectoderm; only the cells of Bolwigs organ
remain in the ectoderm. The ectodermal
region olex is also internalized and forms
an external 'cover' of the optic lobe; many cells
of this population undergo apoptosis (Daniel, 1999).
Epidermal growth factor receptor is activated in midline
regions of the head neurectoderm, in particular in the anlage
of the visual system. Moreover,
increased and/or ectopic activation of Egfr results in a
'cyclops' phenotype very similar to what has been described for
ectopic tll expression. Egfr signaling has been shown to be
required in both chordotonal organs and compound eye
for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated:
(1) is Egfr signaling required for tll expression in the optic
lobe and
(2) is Egfr signaling involved in the recruitment
of the secondary (non-atonal-expressing) Bolwigs
organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for
the presence of Egfr-relevant mRNAs or proteins:
Rhomboid mRNA, which would be expected to be present
only in the signaling cells, and phosphorylated
MAPK, Pointed and Argos mRNAs, which would be
expected to be expressed in all cells receiving an
Egfr-mediated signal. In stage 12 embryos, rho is
expressed only in the small group of Bolwigs organ
founder cells (the same cells expressing ato).
In contrast, activated (phosphorylated) MAPK is
present in a larger cluster of cells including the entire
Bolwigs organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both
known to be switched on in cells receiving the Spi
signal, are expressed at the same stage throughout the
entire Bolwigs organ primordium.
These gene expression and MAPK activation
patterns are consistent with the idea that the Spi signal
is activated by rho in the Bolwigs organ founders and
passed to the neighboring secondary Bolwigs organ
cells where it activates the Egfr signaling cascade.
Supporting this view, only 3-4 photoreceptor neurons
are found in the Bolwigs organ of embryos lacking
rho or spi; furthermore, the size of the
posterior lip of the optic lobe is reduced in such
embryos. The fact that absence of
secondary Bolwigs organ cells in rho or spi mutant
embryos can be rescued by blocking cell death in the
background of a deficiency that takes out the reaper
complex of genes indicates that the Spi signal is not necessary
for the specification (recruitment) of secondary Bolwigs organ
cells, but rather, for their maintenance (Daniel, 1999).
Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitz group genes, yet its underlying cause is unknown. This phenotype has been studied and a new function has been discovered for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes, Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning (Urban, 2004).
The denticle belt fusion phenotype is one of the distinguishing features of the spitz group genes (Mayer, 1988), yet its developmental basis has remained mysterious, since no function has been known for EGFR signalling in the smooth cuticle, which is the affected tissue. Analysis of this phenotype has revealed its cause and uncovered a previously unrecognized function for EGFR signalling in Drosophila epidermal development. Spitz is the primary
EGFR ligand in epidermal patterning, and is activated by proteolysis in three rows of rhomboid-1-expressing cells in the future denticle region. High EGFR signalling is required for cells to adopt the denticle
fate, and other signalling pathways are used to elaborate the different denticle morphologies. The Wingless signal emanates from one posterior row of each parasegment and spreads anteriorly, suppressing the denticle fate and thus allowing cells to secrete a smooth cuticle. These future
smooth-cuticle cells also require signalling through the EGFR for viability, and its absence results in apoptosis of future smooth-cuticle cells and thus denticle belt fusions. This survival signalling is mediated by low-level stimulation of the EGFR by cooperation between the ligands Vein and Spitz, which is activated by Rhomboid-1, Rhomboid-3 and Star (Urban, 2004).
The ventral epidermis is patterned in multiple stages during development, with cell fate specification occurring late, through antagonism between EGFR and Wingless signalling around stages 12-14. Direct phenotypic analysis indicates that EGFR signalling is required for
smooth-cuticle cell survival during these fate specification stages and not earlier or later: epidermal cell apoptosis is greatly elevated in mutant
embryos at stages 12-14, and the fusion phenotype first becomes apparent
around stage 15 as curvature of Engrailed stripes (Urban, 2004).
This direct phenotypic analysis is also supported by several independent genetic observations. EGFR signalling is not required for survival in future smooth-cuticle cells early, when the ventrolateral fates are being specified (stage 10/11) since removing Rhomboid-1 expression at only this stage using the single-minded mutation never results in denticle belt fusions (Mayer, 1988). Defects at this early stage also cause ventral narrowing in spitz group genes (Mayer, 1988), and since rhomboid-3 does not
enhance this phenotype, this suggests that rhomboid-3 cooperates with rhomboid-1 only later in development. Vein acts independently of spitz group genes to suppress denticle belt fusions, and this cannot occur at stage 10/11 since at this early stage Vein expression is dependent on EGFR signalling through a positive feedback loop.
Finally, the fusion phenotype itself suggests that it forms late since
denticle cells are being pulled into smooth cuticle regions and, as such,
their denticle fate must have already been determined and cannot be altered by receiving signals from these smooth domains (Urban, 2004).
Thus, two thresholds with different outcomes exist for EGFR signalling in patterning the ventral epidermis. The level of EGFR signalling that a cell receives is presumably dependent on its distance from the Spitz-processing cells; activated MAPK staining indicates that these rows of cells receive high levels of EGFR signalling. High levels of EGFR signalling are required to induce the
denticle fate, while lower levels that reach smooth-cuticle cells are
sufficient to suppress apoptosis. All ventral epidermal cells therefore
require EGFR signalling, but the exact level, together with antagonism of
shavenbaby transcription by Wingless signalling, determines the
biological outcome. Importantly, these functions may be separate, since Wingless signalling is known to antagonize shavenbaby transcription to repress the denticle fate, but may not repress EGFR signalling itself in smooth-cuticle cells: activated MAPK staining suggests that some
smooth-cuticle cells in the midline may also receive higher levels of EGFR
signalling (Urban, 2004).
These results indicate that cells require EGFR signalling for their survival only when they are starting to differentiate. A similar pattern was also observed in the developing eye imaginal disc where removing the EGFR resulted in cell death only once the morphogenetic furrow had passed. These observations raise the intriguing possibility that
establishing a requirement for survival signals may be inherent in the
differentiation program itself, perhaps for protecting against developmental errors. However, the observation that the requirement for survival signalling is restricted to the central region of the ventral epidermis implies that either this requirement is not ubiquitous, or that another signal is also involved (Urban, 2004).
Pointed is an Ets domain-containing transcription factor that is
responsible for transducing most known instances of EGFR signalling. Although it was previously clear that pointed mutant embryos rarely display denticle belt fusions (Mayer, 1988), analysis of a more recent null allele that removes both P1 and P2 transcripts demonstrates that even complete loss of pointed leads only to a very low frequency of denticle belt fusions. This is also consistent with the milder effects of pointed clones in the developing eye, and in particular the late onset of their apoptosis. These observations raise the possibility that EGFR-mediated survival signalling in general occurs primarily at a non-transcriptional level. Consistent with this model, EGFR signalling has been shown to reduce Hid protein stability, thus directly inhibiting apoptosis (Urban, 2004).
Rhomboid exists as a seven-member family in Drosophila, and at
least four of these members are intramembrane serine proteases that can cleave all
Drosophila membrane-tethered EGFR ligands and specifically activate
EGFR signalling in vivo. Although the precise role of the rhomboid protease family in EGFR signalling and in other biological contexts has been unclear, mutations have now been isolated for both Rhomboid-2 and -3.
Genetic analysis with null alleles has revealed that both act as
tissue-specific activators of EGFR signalling much like Rhomboid-1. Rhomboid-2 is the only rhomboid known to be expressed early in gametogenesis, and is involved in sending EGFR signals from the germline to the soma to guide its encapsidation by somatic cells. In this context, Rhomboid-2 appears to act alone. Rhomboid-3 displays strong expression in the developing eye imaginal disc, and is allelic to roughoid, one of the first Drosophila mutants
described. Rhomboid-3 is the dominant rhomboid protease during eye
development, but does not act alone: Rhomboid-3 cooperates with Rhomboid-1 in the developing eye (Urban, 2004).
Despite the power of these genetic approaches, it should be noted that
rhomboid-1, -2 and -3 exist as a gene cluster on
chromosome 3L and, as such, combined mutations are difficult to generate by recombination. Analysis of epidermal patterning using RNAi to overcome this limitation is the first implication of a rhomboid homolog function in embryogenesis. Interestingly, the rhomboid involved is Rhomboid-3, the
rhomboid that was previously thought to be eye-specific.
However, unlike in the developing eye where Rhomboid-3 has the dominant role, and removing Rhomboid-1 by itself has no effect, the exact opposite is true in embryogenesis: Rhomboid-1 is the main protease in epidermal patterning while removing Rhomboid-3 alone does not result in detectable defects. This analysis suggests that different rhomboid proteases
function predominantly to activate EGFR signalling in distinct tissues, but often act cooperatively or with a degree of redundancy (Urban, 2004).
The requirement for high levels of signalling for fate specification and lower levels for viability in developing tissues may not be limited to the EGFR pathway. Intriguingly, analysis of cell death in wingless mutant embryos suggests that a reciprocal signalling function may also be required to maintain cell viability in denticle regions of the ventral epidermis: in conditions of reduced Wingless signalling, specifically during the stage of epidermal fate specification (but not earlier), cells corresponding to two denticle rows were observed to undergo apoptosis.
Therefore, as with EGFR signalling, high levels of Wingless signalling induce the smooth-cuticle cell fate, while lower levels may be required for survival of a subset of denticle cells. Thus, the Wingless and EGFR signalling pathways may act antagonistically in specifying cell fate, while having complementary and reciprocal functions in maintaining cell viability in the developing epidermis of Drosophila. These survival functions may be conserved since EGFR signalling also has multiple roles in mammalian epidermal development, while some mammalian epidermal tumors are also specifically dependent on EGFR signalling for cell survival. Wnt signalling has also been linked to maintaining cell viability in certain developmental contexts (Urban, 2004).
The Drosophila EGF receptor (Torpedo/DER) and
its ligand (Gurken) play roles in the determination of anterioposterior and dorsoventral axes of the
follicle cells and oocyte. The roles of DER in establishing the polarity of the follicle cells
were examined further, by following the expression of Egfr-target genes. One class of genes
(e.g. kekon) is induced by the Egfr pathway at all stages. Kekon is a novel member of both the leucine-rich repeat and immunoglobulin superfamilies and is a target for Epidermal growth factor signaling in the dorsal-anterior follicle cells and other domains of Egfr signaling. Broad expression of kekon at the
stage in which the follicle cells migrate posteriorly over the oocyte, demonstrates the
capacity of the pathway to pattern all follicle cells except the ventral-most rows. This may
provide the spatial coordinates for the ventral-most follicle cell fates. A second group of
target genes (e.g. rhomboid (rho)) is induced only at later stages of oogenesis, and may
require additional inputs by signals emanating from the anterior, stretch follicle cells. The
function of Rho was analyzed by ectopic expression in the stretch follicle cells, and shown to
induce a non-autonomous dorsalizing activity that is independent of Gurken. Rho thus
appears to be involved in processing an Egfr ligand in the follicle cells, to pattern the egg
chamber and allow persistent activation of the Egfr pathway during formation of the dorsal
appendages (Sapir, 1998).
The expression of Kekon provides a sensitive reporter for Egfr activation, and clarifies the spatial
and temporal aspects of dorsoventral patterning by the pathway. At stages 8-9, when the follicle cells migrate
posteriorly over the oocyte, expression of Kekon is very broad.
This pattern is induced along the two axes. Initially, posterior
migration of the follicle cells over the source of high Gurken
induces activation of the Egfr pathway in all follicle cells
passing over the oocyte nucleus. Next, the lateral diffusion of
the Grk signal leads to a symmetrical lateral activation, which
decreases toward the ventral follicle cells. Consequently,
expression of the marker is induced in all follicle cells, except in the ventral-most rows. This pattern has the capacity to define, by default, the fate of the ventral-most rows of cells (Sapir, 1998).
The next cycle of Egfr activation takes place at stage 10,
when the follicle cells have completed their posterior
migration. The induction of rho expression is critical,
triggered by Gurken-mediated Egfr activation. Rho expression
is essential for dorsoventral patterning, since expression of antisense rho in all follicle cells can lead to the generation of ventralized egg chambers and embryos (Sapir, 1998).
However it is implausible that this dorsoanterior expression domain can define the dorsal and
ventral regions of the egg chamber. What then is the function
of this wave of Egfr activation with respect to dorsoventral
patterning? One possibility is that this phase has an additive effect to the
previous activation of Egfr, which took place during follicle
cell migration. The combined effects of both phases would determine the capacity of the follicle cells to become dorsal. Normally, activation of the Egfr pathway in the Rho-expressing
cells does not seem to extend beyond these cells, as monitored by expression of the Egfr-target gene kekon. It is thus possible that relay mechanisms extend a second, unknown
dorsalizing signal from the Rho-expressing cells to the more
lateral and posterior follicle cells. In conclusion,, Rho-dependent signaling appears to be important
for patterning the dorsal appendages. This is supported by the
persistent expression of kekon and rho in the precursors of the
dorsal appendages until the final stages of oogenesis, and by the induction of multiple dorsal appendages
following ectopic Rho expression. Patterning of the dorsal
appendages may thus represent another distinct Egfr-dependent process (Sapir, 1998).
While the kekon gene is induced at each phase of Egfr
activation, rho expression is triggered only from stage 10
onwards. A mechanism must exist to prevent rho, and possibly
other genes (e.g. bunched), from being
triggered by the same pathway at earlier stages. One option is
that induction by the DER pathway is not sufficient to trigger rho, and an additional input must be provided by a different group of genes from the stretch follicle cells. Dpp is expressed in these cells and may prove to be a likely candidate. Multiple requirements for triggering rho expression may thus
assure that it will normally be induced at a restricted point in space and time, only when the Gurken-induced signal emanating from the oocyte nucleus can be combined with a
signal originating from the stretch follicle cells (Sapir, 1998).
During Drosophila oogenesis Gurken, a TGF-alpha like protein localized close to the oocyte nucleus, activates the
MAPK cascade via the Drosophila EGF receptor (Egfr). Activation of this pathway induces different cell fates in the overlying
follicular epithelium, specifying the two dorsolaterally positioned respiratory appendages and the dorsalmost cells separating
them. Signal-associated internalization of Gurken protein into follicle cells demonstrates that the Gurken signal is spatially
restricted and of constant intensity during mid-oogenesis. Gurken internalization can first be observed in all posterior follicle cells, abutting the oocyte from stage 4 to 6 of oogenesis. At the same time MAPK activation evolves in a spatially and
temporally dynamic way and resolves into a complex pattern that presages the position of the appendages. Therefore, different
dorsal follicle cell fates are not determined by a Gurken morphogen gradient. Instead they are specified by secondary signal
amplification and refinement processes that integrate the Gurken signal with positive and negative feedback mechanisms
generated by target genes of the Egfr pathway (Peri, 1999).
In wildtype egg chambers rhomboid expression pattern can first be detected in a broad domain centered on the anterodorsal corner of the oocyte at the transition from stage 9 to stage 10a of oogenesis, at a time when MAPK activation cannot yet be detected in the follicular epithelium. Thereafter, RHO mRNA starts to be downregulated dorsally, beginning at the anterior margin of the domain. Dowregulation proceeds until the remnants of the first broad expression domain are reduced to a wide ring. rho expression is, at that time, retained only in a small patch in the dorsalmost cells and in two stripes extending laterally towards the nurse cell border. In these cells rho expression is then strongly upregulated and a refinement process begins that leads to a final pattern consisting of two L-shaped domains. Just as rho refinement starts in the dorsalmost part of the egg chamber, MAPK activation in the follicular epithelium first exceeds the detection threshold in exactly the same cells. During the following expansion of the MAPK domain, the strong activation at the leading edge seems to coincide with the refining stripes of rho expression. As the MAPK activation pattern reaches its final spectacle shape, again the dorsal and anterior portions of the MAPK staining pattern are associated with rho expression. In conclusion, rho expression precedes the detection of MAPK activation and both staining patterns are spatially and temporally well correlated. Rhomboid thus may be one of the factors responsible for generating the amplification and modulation of the initial Gurken signal (Peri, 1999).
Gurken signaling also induces the expression of a negative element in the Egfr pathway. Argos expression comes too late to explain the dynamic evolution of the MAPK and rhomboid patterns during stage 10 of oogenesis, but sprouty is expressed early enough to be part of the regulatory network controlling the Egfr pathway activation at stage 10. sprouty is induced in all posterior follicle cells abutting the oocyte at stage 4 to 6 of oogenesis. sty is absent from egg chambers lacking Gurken function, and thus sty may be one factor required to counteract the potential rho-dependent autoactivation of the Egfr pathway (Peri, 1999).
Autocrine signaling through the Epidermal growth factor receptor operates at various stages of
development across species. A recent hypothesis suggested that a distributed network of Egfr autocrine loops is
capable of spatially modulating a simple single-peaked input into a more complex two-peaked signaling pattern,
specifying the formation of a pair organ in Drosophila oogenesis (two respiratory appendages on the eggshell) (Wasserman and Freeman, 1998). To
test this hypothesis, genetic and biochemical information about the Egfr network were integrated into a
mechanistic model of transport and signaling. The model allows the relative spatial ranges and time scales of the relevant feedback
loops to be estimated, the phenotypic transitions in eggshell morphology to be interpreted and the effects of new genetic manipulations to be predicted. It has been found that the
proposed mechanism with a single diffusing inhibitor is sufficient to convert a single-peaked extracellular input into a two-peaked pattern of
intracellular signaling. Based on extensive computational analysis, it is predicted that the same mechanism is capable of generating more complex
patterns. At least indirectly, this can be used to account for more complex eggshell morphologies observed in related fly species. It is proposed that
versatility in signaling mediated by autocrine loops can be systematically explored using experiment-based mechanistic models and their analyses (Shvartsman, 2002).
The mechanism that converts a single-peaked Gurken input into a more complex pattern of MAPK signaling in the responding epithelial cells has been analyzed. Interest in this particular event in Drosophila oogenesis mechanism is twofold. First, it provides an excellent example of versatility in signaling, demonstrating how
simple stimuli define complex patterns in development. More importantly, this
mechanism is arguably one of the best studied at the genetic and biochemical level. It therefore provides a good target for the development of modeling
methodologies and experimental tests of modeling predictions (Shvartsman, 2002).
The oocyte-derived signal is modulated by a network of feedback loops in the follicular epithelium. One positive feedback loop is established when the Egfr, which acts through the Ras/MAPK pathway, induces the expression of rhomboid.
Rhomboid is an intracellular protease that, together with intracellular protein Star, is responsible for processing and secretion of Spitz, another
TGF-beta like ligand of the Egfr. The secreted Spitz directly interacts with the Egfr, further stimulating the intracellular MAPK. Thus, Rhomboid acts as a positive regulator of EGFR signaling. This positive feedback, together with another one mediated by a secreted EGFR ligand Vein, both amplifies and spatially expands MAPK signaling induced by Gurken (Shvartsman, 2002 and references therein).
The amplified and expanded signal is then downregulated by a number of negative feedback loops in the Drosophila Egfr system. Although there are large numbers of negative regulators of Egfr signaling in Drosophila, the original mechanism invokes a single endogenous inhibitor, Argos
(Wasserman, 1998). The expression of argos is induced at high levels of MAPK signaling. Argos is a secreted protein that directly interacts with the
extracellular domain of the Drosophila Egfr, and inhibits intracellular MAPK signaling induced by Gurken, Vein and Spitz. According to Wasserman,
the amplification of the Gurken signal by Spitz and Vein, and its inhibition by Argos first expands the domain of MAPK signaling and then splits it into two smaller
domains, establishing in this way the two groups of appendage-producing cells. In this mechanism, the two peaks detected in the MAPK signaling pattern
(Wasserman, 1998) are closely linked and co-localized with the two stripes that have been repeatedly observed in the pattern or rhomboid
expression (Shvartsman, 2002).
The mechanism of Wasserman links gurken, Egfr, spitz, rhomboid, vein and argos into a system of interconnected feedback loops that are jointly
regulated by the EGFR and intracellular MAPK signaling (Wasserman, 1998). Although the mechanism is supported by genetic and biochemical data,
several important questions remain unanswered. (1) Is the proposed network actually capable of converting single-peaked inputs into persistent two-peaked
outputs? An independent measurement of the MAPK dynamics in oogenesis reports patterns that are more complex, and cannot be as straightforwardly correlated
with the number of appendages of the mature egg. (2) Although the necessary role of argos is supported by genetic approaches, the role of other known Egfr inhibitors remains to be clarified. Argos is the only secreted inhibitor of Egfr in
Drosophila. If it is the only necessary inhibitor, does this mean that the other negative regulators
merely serve to modulate the basic pattern established by the secreted Argos? This must be reconciled with the phenotypic transitions in eggshell morphology induced
by changing the levels of other inhibitors. (3) Several papers have indicated that the expression of argos is detected later than the
relevant changes in the rhomboid expression. Can this be used to argue against the mechanism with a single secreted
inhibitor? To summarize, the sufficiency of the proposed mechanism with a single diffusing inhibitor and its consistency with the large body of genetic data remains to
be established (Shvartsman, 2002).
Based on the computational analysis of this model, the proposed mechanism of pattern formation by peak splitting (Wasserman, 1998) has been validated. The proposed network of positive- and negative-feedback loops in the Egfr system can indeed convert a quasistatic, single-peaked input, into stable, two-peaked outputs. Analysis of the parametric dependence of the stationary patterns in the model indicates that the underlying mechanism accounts for a number of experimentally observed phenotypic transitions (Shvartsman, 2002).
Stationary patterns predicted by the model fall into several qualitatively different classes characterized by the number of peaks in the signaling profiles. The transitions between the classes are discontinuous; this might explain why numerous experiments can be classified in terms of a small number of phenotypes. Stable two-peaked patterns not only exist in this model, but are also kinetically accessible from the state of zero stimulation and are realized through inputs with a single maximum. These stable two-peaked patterns are robust for a wide range of network inputs and strengths of the positive and negative feedback loops. The existence of these stationary patterns requires intermediate input amplitudes and widths, intermediate strengths of the positive feedback, as well as a sufficiently strong and long-ranged inhibition. The fact that large parameter changes induce transitions to qualitatively different patterns is consistent with a large body of genetic data (Shvartsman, 2002).
Several of the predictions of the original mechanism are at variance with experiments. The first quantitative disagreement is observed in the behavior of the two-peaked patterns upon increases in the doses of the stimulatory signal or the strength of the positive feedback. In the model, these changes produce a discontinuous transition to the pattern with a single broad peak; this collapse of the two peaks is preceded by their slight separation. In experiments, eggs laid by the females with extra copies of gurken, heat-shock activated rhomboid, or deficient in Cbl (a negative Egfr regulator) have significantly increased inter-appendage distance. However, a broad appendage phenotype has been reported in a recent experiment that had used tissue-specific gene expression to increase the level of oocyte-derived Gurken (Shvartsman, 2002 and references therein).
A more serious problem is related to the role of argos. First, it is unclear why the onset of argos expression is detected much later than the major changes in the patterns of rhomboid and MAPK activity. The second question, based on the analysis of the model, is related to the relative position in the maxima of rhomboid and argos gene expression patterns. According to the model, the maxima in the expression of the two genes should be co-localized; this is an immediate consequence of the fact that both genes require receptor activity for their production. At the same time, several independent measurements find that for a while argos is expressed between the two regions of high rhomboid expression. If the resulting two-peaked signaling patterns are quasi-stationary, as suggested by the analysis of the original mechanism, then it is unclear what maintains argos expression in the region of decreased MAPK activity. One possibility is that the rate of argos expression is much slower than that of rhomboid, and that the experimentally observed patterns should be interpreted as a transient in the model. This mechanism has been computationally explored and rejected; making the generation of Argos much slower than that of Rhomboid generates pathologic oscillatory instabilities of the two-peaked patterns. Another possible explanation for the observed relative location of the maxima in the gene expression patterns might involve an additional feedback loop. In this extended mechanism, a yet undiscovered positive feedback regulates the expression of argos, making it insensitive to decreases in Egfr signaling after peak splitting (Shvartsman, 2002).
In order for the peak-splitting mechanism to work, the differences in the thresholds of Argos and Rhomboid production must not be too large. In the simulations, the difference in these thresholds is 25%. It has been found that in the case when Argos generation is characterized by a significantly higher threshold than that of Rhomboid, only the one-peaked patterns are realized and peak splitting does not occur. In fact, peak splitting requires a rather delicate balance between the spatially distributed stimulation by Gurken and inhibition by Argos (Shvartsman, 2002).
The analysis supports the original hypothesis about the differences in the spatial ranges of Argos and Spitz. The cause of this separation of length scales is still unclear. Argos and Spitz are secreted into the gap between the oocyte and the follicle cells, where their transport is accompanied by binding to Egfrs in the follicular epithelium. The strength of ligand/receptor binding can regulate the range of the secreted signal in this situation. If this is the case, then the binding constant characterizing the Argos/Egfr interaction should be less than that of Spitz. Surprisingly, it was found that Argos has a higher affinity for the Egfr (but, at the same time, an alternative Argos-like EGF mutant has a lower Egfr-binding affinity). Another mechanism regulating the range of secreted ligands relies on their receptor-mediated endocytosis. Based on the fact that all ligands of mammalian Egfrs are rapidly internalized, it is believed that this mechanism can control spatial ranges of Egfr ligands in Drosophila oogenesis. Furthermore, it is known that Argos interaction with Egfr prevents receptor dimerization and phosphorylation. Since these processes are required to initiate receptor-mediated endocytosis of receptor tyrosine kinases, this further supports the mechanism in which the ranges of Argos and Spitz are controlled by the rates of their internalization (Shvartsman, 2002).
The model does not rely on the difference between the diffusivities of Spitz and Argos. This is in contrast to the classical activator-inhibitor mechanism for morphogenesis proposed by Turing (1952). Instead, it relies on differences of ranges of these molecules, which depend on a combination of their diffusivities and rates of degradation. This is also true for the Turing mechanism; however, in the latter, the difference between the time constants of the activator and the inhibitor leads to oscillatory instabilities. Moreover, the model devised in this study is different from the activator-inhibitor models: while Argos plays the role of a long-range inhibitor, the positive feedback is operated by an autocrine switch with a non-diffusing component (Rhomboid) and a short-ranged diffusing messenger (Spitz). Here, it is the time constant of Spitz degradation that determines the range of the positive feedback; however, the existence of the oscillatory instability is determined by the ratio of the time scales of Rhomboid and Argos. Furthermore, it is not difficult to show that with the given relationship between the thresholds of Rhomboid and Argos production the Turing-like instability (as a result of the homogeneous increase of the level of Gurken input) is not realized at all in the current model. Another difference between this model and that of Turing is that in this model, pattern formation occurs as a result of the instabilities, leading to the abrupt formation and transitions between large-amplitude localized patterns. Large amplitude localized patterns, often referred to as autosolitons, are frequently encountered in different nonlinear systems (Shvartsman, 2002).
In addition to patterns with one and two peaks, the mechanism supports more complex patterns. If the number of peaks in the profile of the receptor activity determines the number of respiratory appendages, then this finding predicts that more complex eggshell phenotypes are to be expected upon quantitative variation of the parameters of the Egfr network. At this point there is a single published observation of eggs with four appendages in mutants of D. melanogaster. Occasionally, eggs with three appendages are laid by the mutants with defects in Gurken accumulation. Notably, eggs with four and even six appendages represent wild-type phenotypes of several related fly species: the eggs of D. virilis have four appendages, while the eggs laid by the flies of subgenus Pholadoris have six appendages. According to the current model, eggs with multiple appendages can be generated by increases in the width and amplitude of the stimulatory signal. Further experimental, modeling and computational studies are required to check whether these more complex phenotypes can be realized in oogenesis or whether they manifest a pathological feature of the mechanism with a single diffusing inhibitor (Shvartsman, 2002).
In Drosophila melanogaster, the patterning of dorsal appendages on the eggshell is strictly controlled by EGFR signaling. However, the number of dorsal appendages is remarkably diverse among Drosophila species. For example, D. melanogaster and D. virilis have two and four dorsal appendages, respectively. During oogenesis, the expression patterns of rhomboid (rho) and argos (aos), positive and negative regulators of EGFR signaling, respectively, are substantially different between D. melanogaster and D. virilis. Importantly, the number of locations and position of both rho expression and MAPK activation are consistent with those of the dorsal appendages in each species. Despite the differences in spatial expression, these results suggest that the function of EGFR signaling in dorsal appendage formation is largely conserved between these two species. Thus, these results link the species-specific activation of EGFR signaling and the evolution of eggshell morphology in Drosophila (Nakamura, 2003).
The twin-peaks model for D. melanogaster dorsal appendage induction suggests that initial EGFR activation is triggered by Grk localized at the dorsal midline of the oocyte at stage 8/9. This leads to the expression of rho in the overlying follicle cells that receive the Grk signal. In cooperation with Spi, Rho triggers the positive feedback loop of EGFR signaling at stage 10. Consequently, the EGFR signal reaches its highest level at the dorsal midline, leading to the subsequent induction of aos at the dorsal midline at stage 11. The resulting signaling profile has twin peaks that maintain rho expression as a stripe of cells on either side of the dorsal midline (Nakamura, 2003 and references therein).
Surprisingly, although MAPK activation was detected in the midline in D. virilis, the initial expression of rho was detected in two groups of follicle cells on the dorsolateral sides. Notably, no rho expression was detected in the dorsal midline. Furthermore, aos expression was not observed in the dorsal midline from stage 10 to 11, which suggests that Aos is not involved in inhibiting the EGFR signal in the dorsal midline. This observation suggests that in D. virilis the stripe of rho-expressing cells on either side of the midline is not specified by the sequential induction of rho and aos, in contrast to the twin-peaks model in D. melanogaster. Based on these results, it is speculated that a negative regulator of EGFR signaling, other than Aos, is induced primarily in response to the Grk signal, and leads to the expression of rho in two domains in each lateral half. It is speculated that a low level of EGFR signaling is sufficient to induce this putative inhibitor(s), and that this inhibitor(s) is induced in the broad area of the dorsoanterior region, because MAPK activation and rho expression are excluded from a broader area of this region in D. virilis than in D. melanogaster. This putative negative regulator(s) of EGFR signaling is probably induced prior to the induction of aos. Thus, aos may never be induced in the dorsal midline in D. virilis, because EGFR signaling never reaches a threshold that is high enough to induce it in this scenario. From studies in D. melanogaster, kekkon-1 and sprouty, two negative regulators of EGFR signaling, are strong candidates, although this issue has to be addressed in D. virilis. Alternatively, an enhancer of rho may gain a novel characteristic to be repressed in response to the high level of EGFR signaling (Nakamura, 2003).
The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals, results supporting this hypothesis were obtained. Tests were performed for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, it was found that both EcR-B isoforms are able to rescue the neuronal defect cell autonomously, but EcR-A is less effective. The effect of over-expressing the isoforms was examined in a wild-type background. A sensitive period was determined when high levels of either EcR-B isoform are lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast has no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppress puparial tanning, and result in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect (Schubiger, 2003).
Loss of function of both EcR-B isoforms inhibits dendritic pruning by the larval Tv-neurons, a set of thoracic FMRFamide expressing neurons. However, since all cells in these animals are mutant, it was not known if the failure to prune dendrites was a cell autonomous defect. Consequently, it was asked if the dendritic pruning defect could be rescued by expressing the different EcR isoforms specifically in these neurons. Using the FG5-Gal4 driver containing regulatory sequences of the FMRFamide gene wild-type EcR isoforms as well as the GFP-labeled membrane marker mCD8 were expressed in EcR-B mutant animals. The cell-autonomous rescue of pruning in the Tv neurons shows that given the correct set of receptors in these neurons, ecdysone can induce a pruning response even in a mutant environment in which overall nervous system development is arrested. Both the pruning of these neurosecretory cells and the pruning of the mushroom body neurons require the presence of an EcR-B isoform. The pruning of the Tv-cells, however, is only inhibited when both EcR-B1 and EcR-B2 are absent. This differs from the mushroom body neurons that fail to undergo pruning when only EcR-B1 is missing. Nevertheless similarity across different neuronal types suggests that a general set of rules may be employed early in metamorphosis to remove larval specializations (Schubiger, 2003).
The Drosophila egg chamber provides an excellent model for studying
the link between patterning and morphogenesis. Late in oogenesis, a portion of
the flat follicular epithelium remodels to form two tubes; secretion of eggshell
proteins into the tube lumens creates the dorsal appendages. Two distinct cell
types contribute to dorsal appendage formation: cells expressing the
rhomboid-lacZ (rho-lacZ) marker form the ventral floor of the tube
and cells expressing high levels of the transcription factor Broad form a roof
over the rho-lacZ cells. In mutants that produce defective dorsal
appendages (K10, Ras and ectopic decapentaplegic) both cell
types are specified and reorganize to occupy their stereotypical locations
within the otherwise defective tubes. Although the rho-lacZ and Broad
cells rearrange to form a tube in wild type and mutant egg chambers, they never
intermingle, suggesting that a boundary exists that prevents mixing between
these two cell types. Consistent with this hypothesis, the Broad and
rho-lacZ cells express different levels of the homophilic adhesion
molecule Fasciclin 3. Furthermore, in the anterior of the egg, ectopic
rhomboid is sufficient to induce both cell types, which reorganize
appropriately to form an ectopic tube. It is proposed that signaling across a
boundary separating the rho-lacZ and Broad cells choreographs the cell
shape-changes and rearrangements necessary to transform an initially flat
epithelium into a tube (Ward, 2005).
Each dorsal appendage is made from a population of cells that reorganizes
from an initially flat epithelium into a tube. This process most closely
resembles wrapping, one of a variety of mechanisms that produce epithelial tubes. Dorsal
appendage tube formation exhibits all three characteristics of the wrapping
mechanism. (1) The dorsal appendage cells maintain epithelial contacts during
tube formation.
(2) The Broad cells constrict apically, causing the flat epithelium to curve.
(3) The dorsal appendage tubes are parallel to the follicular epithelium, rather than
perpendicular as observed in budding tubes. These features also characterize
vertebrate neural tube and Drosophila ventral furrow formation (Ward, 2005).
Dorsal
appendage tube formation differs from neural tube/ventral furrow formation in
one key respect. The neural tubes and ventral furrow are each made by a
symmetric fold in the epithelium. In contrast, asymmetric shape-changes and
movements produce each dorsal appendage tube. Prior to tube formation, the
rho-lacZ rows are perpendicular to one another in a pattern resembling an
open hinge. Then, during tube formation the anterior row of rho-lacZ
cells moves posterior, thereby closing off the ventral midline of the dorsal
appendage tube. Cells in the medial (dorsal) row elongate concomitant with the
apical constriction of the roof cells, but these floor cells do not swing
anteriorly. Thus, during dorsal appendage formation the perpendicular rows of
rho-lacZ cells do not move equivalent distances to seal the tube.
Evidently, this process is both robust and malleable, since normal tubes can still
form in patterning mutants with altered primordia (Ward, 2005).
What mechanism ensures
proper tube closure? Recall, during tube formation the Broad pattern
simultaneously shortens and lengthens along two perpendicular axes via likely
convergent-extension rearrangements. Since the rho-lacZ and Broad cells
maintain epithelial contacts with one another during tube formation, it is proposed
that the rearrangements among the Broad cells contribute to a reorganization of
the adjacent, underlying rho-lacZ cells. Thus, the anterior-medial
movement of the Broad cells simultaneously lengthens the tube and draws the
anterior row of rho-lacZ cells posteriorly. This process allows the
rho-lacZ cells on either side of the hinge to associate with one another
in a pair-wise fashion to close off the ventral midline of the tube. Similarly,
convergent extension during neural tube development narrows the distance between
the neural folds, allowing them to meet and fuse (Ward, 2005).
Additional insight into patterning and morphogenesis is provided
by analysis of loss of function (Ras1 and K10) and gain of
function (UAS-dpp and UAS-rho) mutants. In each
mutant, the rho-lacZ and Broad cell types are specified and occupy their
stereotypical locations within the otherwise defective tubes. Four features of
these aberrant tubes are noteworthy. (1) The number of cells contributing to
each primordium can vary widely, from a few cells in the UAS-rho clones
to as many as hundreds in K10 egg chambers. Nevertheless, the
rho-lacZ and Broad cells coordinate their movements to form a tube.
(2) The position of the primordium within the egg chamber is not restricted
to the dorsal anterior. Dorsal appendages shift posteriorly when dpp
expression is greatly expanded, and ventral/lateral tubes may form when
UAS-rho is expressed in the collar. Apparently, as long as both cell
types form, other factors are not limiting in tube formation. (3) The
posterior and ventral limits of both rho-lacZ and Broad expression
precisely mirror one another. Even when ectopic rhomboid enlarges the
normal domain of the dorsal appendage primordium, Broad and rho-lacZ
expression expand coordinately. These results suggest that the patterning of
these two cell types is a linked process. (4) In some K10 egg
chambers and in the Ras1 hypomorphs and UAS-rho mutants, the
rho-lacZ cells flank only the anterior margin of each Broad domain.
Although the rho-lacZ cells are not arranged in a hinge pattern, the
dorsal appendage cells reorganize appropriately to form a tube. Thus, the hinge
pattern of rho-lacZ cells is not essential for tube formation (Ward, 2005).
These
results indicate that the juxtaposition of rho-lacZ and Broad cells is
necessary for tube formation and suggests that communication between the two
cell types promotes the cell shape changes and rearrangements necessary to make
a tube from an initially flat epithelium (Ward, 2005).
How do the
dorsal appendage cells reorganize in a coordinated manner to form a tube? It
is proposed that the rho-lacZ and Broad cells are separated by a
'boundary' and that signaling across this boundary choreographs
the cell shape-changes and rearrangements necessary to make a tube from an
initially flat epithelium. Boundaries between two different cell types occur
frequently in developing tissues. Importantly, cells on one side of
a boundary are free to mix with one another, but do not mix with cells on the
other side of the boundary. This fence-like property of boundaries may be
maintained by differences in cell adhesion. Although this hypothesis provides a
satisfying explanation for cell behaviors, the adhesive mechanisms that prevent
intermingling between different cell types are not understood. Finally, a boundary can
function as an ‘organizer’ to instruct cells about their position
and fate within a developing tissue (Ward, 2005).
Boundaries are of two types: lineage
restricted (compartment) and non-lineage restricted. Since the patterning
processes that define the dorsal appendage primorida occur after the cessation
of cell division, a boundary between the rho-lacZ and Broad cells would
be of the non-lineage-restricted type. Previous researchers have proposed that
another boundary exists in the dorsal anterior follicle cells. This boundary is
established by differential Bunched activity and lies between
operculum/non-operculum cells. The boundary described in this paper is between the
rho-lacZ and Broad cells (Ward, 2005).
What is the evidence that a boundary
separates the rho-lacZ and Broad cells? (1) The roof and floor cells
express unique cell-fate markers, display differential levels of cell-adhesion
proteins, and exhibit distinct behaviors such as directed elongation and
convergence/extension. Clearly they are different cell types. (2) Throughout
the elaborate cell shape-changes and rearrangements of dorsal-appendage
morphogenesis, the rho-lacZ and Broad cells coordinate their behaviors
and never intermingle, even when patterning goes awry. Finally, the membrane(s)
between the rho-lacZ and Broad cells accumulates high levels of
phosphorylated proteins, consistent with signaling between the two cell types (Ward, 2005).
Signaling via an organizer established at the boundary may direct
the cell shape-changes and rearrangements necessary to make a tube, perhaps by
instructing the rho-lacZ cells to elongate and the Broad cells to
constrict apically. The boundary could also direct the convergent-extension
rearrangements of the Broad cells. Consistent with an organizer acting at the
boundary between the rho-lacZ and Broad cells, ectopic expression of
rhomboid in the anterior of the egg chamber produces an ectopic boundary
capable of reorganizing the rho-lacZ and Broad cells into a tube. Domains
of high-Broad-expressing cells merely produced warts, whereas clusters of cells
containing both the Broad and rho-lacZ cell types reorganized properly
and synthesized dorsal appendage tubes (Ward, 2005).
Altogether, these wild type, mutant, and
ectopic rhomboid studies indicate that the juxtaposition of
rho-lacZ and Broad cells is necessary to make a dorsal appendage tube.
These two sub-populations of cells express many different cell-fate and adhesion
markers, exhibit distinct behaviors, and never intermingle. It is hypothesized that
a boundary exists between these two cell types and that signaling across the
boundary coordinates the cell shape-changes and rearrangements that form the
tube. These studies offer insight into the processes that regulate tubulogenesis,
reveal mechanistic links between patterning and morphogenesis, and provide a
foundation for inquiry in other systems (Ward, 2005).
Boundaries establish and maintain separate populations of cells critical for organ formation. Notch signaling establishes the boundary between two types of post-mitotic epithelial cells, the Rhomboid- and the Broad-positive cells. These cells will undergo morphogenetic movements to generate the two sides of a simple organ, the dorsal appendage tube of the Drosophila egg chamber. The boundary forms due to a difference in Notch levels in adjacent cells. The Notch expression pattern mimics the boundary; Notch levels are high in Rhomboid cells and low in Broad cells. Notch− mutant clones generate an ectopic boundary: ectopic Rhomboid cells arise in Notch+ cells adjacent to the Notch− mutant cells but not further away from the clonal border. Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression. It is further shown that Broad represses rhomboid cell autonomously. These data provide a foundation for understanding how a single row of Rhomboid cells arises adjacent to the Broad cells in the dorsal appendage primordia. Generating a boundary by the Notch pathway might constitute an evolutionarily conserved first step during organ formation in many tissues (Ward, 2006).
At the boundary, cells with high Notch express rhomboid, whereas cells with lower Notch express Broad. A new boundary is established at Notch− mutant clone borders, where Notch+ cells adjacent to Notch− cells ectopically express rhomboid and do not express Broad. Thus, in the dorsal anterior, when two cells with different Notch levels are adjacent to one another, the cell with higher Notch levels simultaneously represses Broad and promotes rhomboid expression. broad− cells ectopically express rhomboid, indicating that Broad normally represses rhomboid expression. It is inferred that cells with higher Notch levels repress Broad, thereby allowing rhomboid expression. It is now proposed that when cells with different levels of Notch are located next to each other, the cells with high Notch repress Broad, allowing rhomboid expression. In contrast, cells with low Notch express Broad and therefore repress rhomboid expression (Ward, 2006).
Notch, an important modulator of boundary function in other tissues, establishes the boundary that defines the Rhomboid and the Broad dorsal appendage cell types. When Notch is removed from cells that should span the boundary, rhomboid is not expressed, and Broad is ectopically expressed. Thus, at the boundary, Notch regulates the patterning of both Rhomboid and Broad cell types. When Notch activity is removed from Region 1, ectopic Rhomboid cells (Notch+) arise adjacent to Notch− (Broad) cells, thus resembling the normal Notch border. It is proposed that these Notch− mutant clones produce ectopic borders of differential Notch activity, which in turn generate ectopic boundaries between Rhomboid and Broad domains (Ward, 2006).
Normally, Rhomboid cells arise all along the high–low Notch boundary in each dorsal appendage primordium. Based upon this observation, one might expect that Rhomboid cells would surround the Notch− clones. In the current studies, however, it was found that only those cells close to the normal boundary turned on ectopic rhomboid. Two factors probably contribute to this result. First, other signaling pathways, most notably EGFR and DPP, are involved in specifying and positioning the Rhomboid and Broad cell populations within the follicular epithelium. Presumably, these other signaling pathways influence Broad/rhomboid expression in cells adjacent to Notch− clones. Second, the ectopic Notch borders generated by Notch− clones arise within the Broad domain, which normally has low levels of Notch. Therefore, many cells at the ectopic border may not have sufficient Notch activity to repress Broad and activate rhomboid (Ward, 2006).
Within the domain that would normally express Broad, loss of Notch causes the loss of Broad non-cell autonomously in adjacent cells and the appearance of ectopic rhomboid in these same cells. Furthermore, Notch− clones spanning the boundary ectopically express Broad and do not express rhomboid. These findings are consistent with previous results demonstrating that dorsal appendage cells express either rhomboid or Broad, but never both markers. This work shows that broad− cells ectopically express rhomboid, suggesting that one function of Broad in the follicular epithelium is to directly or indirectly repress rhomboid expression. Such regulation must occur (at least in part) in the 2.2-kb fragment that drives lacZ expression in a reporter construct. CONSITE software detects twenty Broad binding sites clustered together in this region; all four zinc-finger isoforms have the potential to bind. Thus, high levels of Broad could directly regulate rhomboid in Region 1. Additional work is needed to test this hypothesis (Ward, 2006).
Other factors must also regulate rhomboid expression in Region 2. Within clones spanning the boundary, ectopic expression of Broad prevents rhomboid expression. In cells adjacent to Notch clones, loss of Broad expression allows ectopic rhomboid expression. Nevertheless, the simple absence of Broad is insufficient to induce rhomboid expression, since the majority of cells in Region 2 lack Broad expression and do not express rhomboid. Presumably, high levels of EGFR and DPP signaling prevent rhomboid expression in these cells (Ward, 2006).
The Notch loss- and gain-of-function data, as well as the Notch expression pattern, all suggest that juxtaposition of two cells with different Notch levels is critical for establishing the boundary between Rhomboid and Broad cell types. How, then, is Notch protein level regulated? The restricted pattern of Notch in the dorsal anterior follicle cells suggests that Notch expression is determined by a combination of patterning instructions from DPP along the anterior/posterior axis and EGFR signaling along the dorsal/ventral axis (Ward, 2006).
The importance of regulating Notch protein levels is underscored by data showing that overexpression of full-length Notch represses Broad expression throughout the follicular epithelium. Since the full-length Notch receptor must be bound by ligand to initiate Notch signaling, a Notch ligand is either present throughout the follicular epithelium or is presented to the follicle cells by the underlying germ line. The Drosophila genome encodes two known Notch ligands, Delta and Serrate, and several potential ligands, such as CG9138. The absence of both Delta and Serrate in the follicular layer did not affect Broad or rhomboid expression. The function of other potential ligands in follicle cells is not currently known. It is also possible that the ligand for this process is present in the germ line. Delta is expressed in the germ line at the appropriate time and functions in the germ line to regulate follicle cell processes, such as the pinching-off of egg chambers in the germarium and the mitotic-to-endocycle transition at stage 7. Additionally, previous work demonstrates that egghead and brainiac, which encode modulators of Notch function, act in the germ line to pattern the dorsal anterior follicle cells. Regardless of the tissue distribution of the ligand, however, the ability to uniformly activate the Notch pathway throughout the follicle cell layer is note-worthy. This observation suggests that Notch levels, rather than spatial location of a ligand (or ligand modulator), determines where or how Notch signals in follicle cells of late stage egg chambers (Ward, 2006).
One of the most surprising aspects of the work presented here is that Notch clones act in a non-cell-autonomous manner to regulate Broad and rhomboid expression in adjacent cells. While surprising, non-cell-autonomous Notch activity occurs in the embryo, and most notably, at the D/V boundary in the wing disc. In the third-instar wing disc, Wingless is expressed in a 3- to 6-cell wide stripe spanning the D/V boundary, which separates the dorsal and ventral portions of the future wing blade. In this system, wingless-lacZ is repressed both within and adjacent to Notch− clones. Thus, Notch− clones act non-cell autonomously in two different tissues where boundaries act to distinguish different cell types (Ward, 2006).
What is the nature of the non-autonomous signal from the Notch− clones? It is proposed two potential mechanisms to explain this process. First, Notch itself measures Notch levels in adjacent cells, either directly through homophilic adhesion or indirectly through interaction with Notch-binding proteins. When a Notch− clone occurs in the dorsal anterior, adjacent cells sense the absence of Notch and respond as wild-type cells do when high-Notch cells neighbor low-Notch cells; they either repress Broad directly, or they repress Broad indirectly by affecting Pangolin (or some other component of the Wingless signaling pathway). Pangolin is needed to express Broad and therefore down-regulate rhomboid throughout the follicle cell layer. A second possibility is that when cells have little or no Notch activity, they might secrete an inhibitor of the Pangolin pathway that only affects cells with high Notch. The first mechanism is favored for its simplicity in accounting for rhomboid expression only at the border between high- and low-Notch-expressing cells (Ward, 2006).
The establishment of a border between Rhomboid and Broad cells is important for preventing intermingling of these cell types during tube formation (Ward, 2005). It is not clear, however, what mechanism separates the Broad and Rhomboid cells from each other at the border. In some situations, the non-transcriptional branch of the Notch pathway regulates F-actin (Major, 2005), which creates a “fence” that could help separate the two cell types from each other in the border. In dorsal anterior follicle cells, however, the canonical Notch pathway acts through the transcription factor Su(H). It is possible that in this cell type, the Notch pathway transcriptionally regulates a cell adhesion molecule or other component of an actin-binding protein complex, which in turn coordinates the cytoskeleton, thereby maintaining a separation between the Rhomboid cells and the Broad cells. Unlike cells at other boundaries in which an actin fence is evident, the Rhomboid and Broad cells undergo dramatic morphological changes and reorganize their actin networks to produce these effects. A fence that could maintain the separation of these cells during apical constriction, directed elongation, and convergent extension would be critical during these processes. One such Notch-interacting candidate gene that links to actin filaments is Echinoid. Future experiments will define whether Echinoid plays a role during border formation between Rhomboid and Broad cells (Ward, 2006).
Animals have a wide variety of organs containing different cell types arranged in a stereotypical manner. While the general morphogenesis of most organs has been described, little is known about the molecular mechanisms required to specify boundaries between diverse cell types and direct their subsequent reorganization to produce a functional structure. This study has shown that canonical Notch signaling is necessary to establish a boundary between the Broad and Rhomboid cells, which will form the dorsal and ventral portions of the dorsal appendage tube. Notch is also required in the vertebrate hindbrain for rhombomere boundary formation. Thus, in simple and more complex organs, Notch specifies boundaries between distinct cell populations needed for organ formation. Generating a boundary through Notch signaling could be an evolutionarily conserved first step during organ formation in many tissues. The next challenge is to define the molecular nature of the physical power that keeps the two different cell types separated from each other in the border (Ward, 2006).
Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Analysis of a large number of published gene expression patterns in the follicle cells suggests that they follow a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of inductive signals, provided by the highly conserved epidermal growth factor receptor and bone morphogenetic protein signaling pathways. The validity of the code is demonstrated by testing it against a set of patterns obtained in a large-scale transcriptional profiling experiment. Using the proposed code, 36 distinct patterns were distinguished for 81 genes expressed in the follicular epithelium, and their joint dynamics were characterize over four stages of oogenesis. The proposed combinatorial framework allows systematic analysis of the diversity and dynamics of two-dimensional transcriptional patterns and guides future studies of gene regulation (Yakoby, 2008b).
Drosophila eggshell is a highly patterned three-dimensional structure that is derived from the follicular epithelium in the developing egg chamber. The dorsal-anterior structures of the eggshell, including the dorsal appendages and operculum, are formed by the region of the follicular epithelium, which is patterned by the highly conserved epidermal growth factor receptor (EGFR) and bone morphogenetic protein (BMP) signaling pathways. The EGFR pathway is activated by Gurken (GRK), a transforming growth factor α-like ligand secreted by the oocyte. The BMP pathway is activated by Decapentaplegic (DPP), a BMP2/4-type ligand secreted by the follicle cells stretched over the nurse cells (Yakoby, 2008b).
Acting through their uniformly expressed receptors, these ligands establish the dorsoventral and anteroposterior gradients of EGFR and DPP signaling and control the expression of multiple genes in the follicular epithelium. Under their action, the expression of a Zn finger transcription factor, Broad (BR), evolves into a pattern with two patches on either side of the dorsal midline. The BR-expressing cells form the roof (upper part) of the dorsal appendages. Adjacent to the BR-expressing cells are two stripes of cells that express rhomboid (rho), a gene that is directly repressed by BR and encodes ligand-processing protease in the EGFR pathway. These cells form the floor (lower part) of the appendages (Yakoby, 2008b).
The patterns of genes expressed during the stages of egg development that correspond to appendage morphogenesis are very diverse. At the same time, inspection of a large number of published patterns suggests that they can be 'constructed' from a small number of building blocks. For instance, the T-shaped pattern of CG3074 is similar to the domain 'missing' in the early pattern of br, while the two patches in the late pattern of br appear to correspond to the two 'holes' in the expression of 18w. Based on a number of similar observations, it was hypothesized that all of the published patterns could be constructed from just six basic shapes, or primitives, which reflect the anatomy of the egg chamber and the spatial structure of the patterning signals (Yakoby, 2008b).
In computer graphics, representation of geometrical objects in terms of a small number of building blocks is known under the name of constructive solid geometry, which provides a way to describe complex shapes in terms of just a few parameters -- the types of the building blocks, such as cylinders, spheres, and cubes, their sizes, and operations, such as difference, union, and intersection. Thus, information about a large number of structures can be stored in a compact form of statements that contain information about the types of the building blocks and the operations from which these structures were assembled. This study describes a similar approach for two-dimensional patterns and demonstrate how it enables the synthesis, comparison, and analysis of gene expression at the tissue scale (Yakoby, 2008b).
The six building blocks used in the annotation system can be related to the structure of the egg chamber and the spatial distribution of the EGFR and DPP signals. The first primitive, M (for 'midline'), is related to the EGFR signal. It reflects high levels of EGFR activation and has a concave boundary, which can be related to the spatial pattern of GRK secretion from the oocyte. The second primitive, denoted by D (for 'dorsal'), reflects the intermediate levels of EGFR signaling during the early phase of EGFR activation by GRK, and is defined as a region of the follicular epithelium that is bounded by a level set (line of constant value) of the dorsoventral (DV) profile of EGFR activation. The boundary of this shape is convex and can be extracted from the experimentally validated computational model of the GRK gradient. The third primitive, denoted by A (for 'anterior'), is an anterior stripe which is obtained from a level set of the early pattern of DPP signaling in the follicular epithelium. This pattern is uniform along the DV axis, as visualized by the spatial pattern of phosphorylated MAD (P-MAD). Thus, the D, M, and A primitives represent the spatial distribution of the inductive signals at the stage of eggshell patterning when the EGFR and DPP pathways act as independent AP and DV gradients (Yakoby, 2008b).
Each of the next two primitives, denoted by R (for 'roof') and F (for 'floor'), is composed of two identical regions, shaped as the respective expression domains of br and rho, and reflect spatial and temporal integration of the EGFR and DPP pathways in later stages of eggshell patterning. The mechanisms responsible for the emergence of the F and R domains are not fully understood. It has been shown that the R domain is established as a result of sequential action of the feedforward and feedback loops within the EGFR and DPP pathways. The formation of the F domain requires the activating EGFR signal and repressive BR signal, expressed in the R domain. Thus, at the current level of understanding, the R and F domains should be viewed as just two of the shapes that are commonly seen in the two-dimensional expression patterns in the follicular epithelium. The sixth primitive, U (for 'uniform'), is spatially uniform and will be used in combination with other primitives to generate more complex patterns (Yakoby, 2008b).
While a number of patterns, such as those of jar and Dad, can be described with just a single primitive, more complex patterns are constructed combinatorially, using the operations of intersection (∩), difference ( ), and union (∪) For example, the dorsal anterior stripe of argos expression is obtained as an intersection of the A and D primitives (A∩D). The ventral pattern of pip is obtained as a difference of the U and D primitives (U D). The pattern of 18w is constructed from the A, D, and R primitives, joined by the operations of union and difference (A∪D R). For a small number of published patterns, the annotations reflect the experimentally demonstrated regulatory connections. For example, the U D annotation for pip reflects that actual repression of pip by the dorsal gradient of EGFR activation. For a majority of genes, the annotations should be viewed as a way to schematically represent a two-dimensional pattern and as a hypothetical description of regulation (Yakoby, 2008b).
The geometric operations of intersection, difference, and union can be implemented by the Boolean operations performed at the regulatory regions of individual genes. Boolean operations evaluate expression at each point and assign a value of 0 (off) or 1 (on). As an example, consider a regulatory module, hypothesized for argos, that performs a logical AND operation on two inputs: the output of the module is 1 only when both inputs are present. When both of the inputs are spatially distributed, the output is nonzero only in those regions of space where both inputs are present, leading to an output that corresponds to the intersection of the two inputs. Similarly, a spatial difference of the two inputs can be realized by a regulatory module that performs the ANDN (ANDNOT) operation. This is the case for pip, repressed by the DV gradient of GRK signaling and activated by a still unknown uniform signal. Finally, a regulatory module that performs an OR operation is nonzero when at least one of the inputs is nonzero. When the inputs are spatially distributed, the output is their spatial union (Yakoby, 2008b).
Boolean operations on primitives lead to patterns with just two levels of expression (the gene is either expressed or not). In addition to Boolean logic, developmental cis-regulatory modules and systems for posttranscriptional control of gene expression can perform analog operations, leading to multiple nonzero levels of output. Consider a module that adds the two binary inputs, shaped as the primitives. The output is nonzero in the domain shaped as the union of the two primitives, but is characterized by two nonzero levels of expression. This type of annotation is reserved only for those cases where the application of Boolean operations would lead to a loss of the spatial structure of the pattern (such as the A + U expression pattern of mia at stage 11 of oogenesis. For example, the union of the A and U primitives is a U primitive, whereas the sum of these primitives is an anterior band superimposed on top of a spatially uniform background (Yakoby, 2008b).
Signaling pathways guide organogenesis through the spatial and temporal control of gene expression. While the identities of genes controlled by any given signal can be identified using a combination of genetic and transcriptional profiling techniques, systematic analysis of the diversity of induced patterns requires a formal approach for pattern quantification, categorization, and comparison. Multiplex detection of gene expression, which has a potential to convert images of the spatial distribution of transcripts into a vector format preferred by a majority of statistical methods, is currently feasible only for a small number of genes and systems with simple anatomies. This paper presents an alternative approach based on the combinatorial construction of patterns from simple building blocks (Yakoby, 2008b).
In general, the building blocks can be identified as shapes that are overrepresented in a large set of experimentally collected gene expression patterns. This approach can be potentially pursued in systems where mechanisms of pattern formation are yet to be explored. At the same time, in well-studied systems, the building blocks can be linked to identified patterning mechanisms. This study chose six primitives based on the features that are commonly observed in real patterns and related to the structure of the tissue as well as the spatial distribution of the inductive signals. A similar approach will be useful whenever a two-dimensional cellular layer is patterned by a small number of signals, when cells can convert smoothly varying signals into spatial patterns with sharp boundaries, and when the regulatory regions of target genes have the ability to combinatorially process the inductive signals. One system in which this approach could be feasible is the wing imaginal disc, which is patterned by the spatially orthogonal wingless and DPP morphogens (Yakoby, 2008b).
The six primitives are sufficient to describe the experimentally observed patterns during stages 10-12 of oogenesis. A natural question is whether it is possible to accomplish this with a smaller number of primitives. Two of the primitives, R and F, could be potentially constructed from the D, M, and A primitives, which are related to the patterns EGFR and DPP activation during the earlier stages of eggshell patterning. Specifically, recent studies of br regulation suggest that the R domain is formed as a difference of the D, A, and M patterns (Yakoby, 2008a). Furthermore, the formation of the F domain requires repressive action in the adjacent R domain. With the R and F domains related to the other four primitives, the size of the spatial alphabet will be reduced even further (from six to four), but at the expense of increasing the complexity of the expressions used to describe various spatial patterns (Yakoby, 2008b).
Previously, the question of the diversity of the spatial patterns has been addressed only in one-dimensional systems. For example, transcriptional responses to the Dorsal morphogen gradient in the early Drosophila embryo give rise to three types of patterns in the form of the dorsal, lateral, and ventral bands. This work provides an attempt to characterize the diversity and dynamics of two-dimensional patterns. Thirty-six qualitatively different patterns were constructed, and it is proposed that each of them can be constructed using a compact combinatorial code. The sizes of the data sets from the literature and from transcriptional profiling experiments are approximately the same (117 and 96 patterns, respectively. Based on this observation, it is expected that discovered patterns will be readily described using this annotation system (Yakoby, 2008b).
A gene expressed in more than one stage of oogenesis is more likely to appear in different patterns, and it was found that groups of genes sharing the same pattern at one time point are more likely to scatter in the future than to stay together. More detailed understanding of the dynamics of the spatial patterns of the EGFR and DPP pathway activation is crucial for explaining these trends and the two observed scenarios for the emergence of complex patterns. A gene that makes its first appearance as a complex pattern, such as the A∩D pattern of argos at stage 10B, can be a direct target of the EGFR and DPP signal integration. In contrast, a gene such as Cct1, which changes from the A to the R pattern, can be a dedicated target of DPP signaling alone, and changes as a consequence of change in the spatial pattern of DPP signaling. Future tests of such hypotheses require analysis of cis-regulatory modules responsible for gene regulation in the follicular epithelium. While only a few enhancers have been identified at this time, this categorization of patterns should accelerate the identification of enhancers for a large number of genes (Yakoby, 2008b).
Proposed for the spatial patterns of transcripts, these annotations can also describe patterns of protein expression, modification, and subcellular localization. For example, the stage 10A patterns of MAD phosphorylation and Capicua nuclear localization can be accurately described using the A and U D annotations, respectively. The ultimate challenge is to use the information about the patterning of the follicular epithelium to explore how it is transformed into the three-dimensional eggshell. A number of genes in the assembled database encode cytoskeleton and cell adhesion molecules, suggesting that they provide a link between patterning and morphogenesis. It is hypothesized that the highly correlated expression patterns of these genes give rise to the spatial patterns of force generation and mechanical properties of cells that eventually transform the follicular epithelium into a three-dimensional eggshell (Yakoby, 2008b).
Continued Rhomboid Developmental Biology part 2/2
rhomboid:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
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