eyegone
Based on its pattern of expression, eyegone is thought to play a role in salivary gland
organogenesis. Salivary gland primordium (SGP) development responds to positional information. On the anteroposterior axis, Sex
combs reduced (Scr) specifies parasegment 2. In Scr minus embryos, no salivary glands are formed and eyg
expression is lost, except for a small patch of cells present at the PS1/PS2 border. In a teashirt minus mutation, Scr is expanded to both PS2 and PS3 and results in enlarged SGPs. The SGP
expression of eyg is duplicated in PS3, although its appearance and fading are delayed slightly.
Along the dorsoventral axis, the SGP is restricted dorsally by decapentaplegic (dpp), and ventrally by the spitz group
of genes. In dpp minus embryos, eyg expression expands dorsally to form a
ring that is interrupted ventrally. In several spitz-group mutant embryos, such as single minded
(sim), the SGPs from each side move ventrally, and eyg expression expands ventrally. Expression
in the trunk is also disordered, which may be a secondary effect of the disruption of the mesoderm (Jun, 1998).
Salivary eyegone expression is regulated positively by Sex combs reduced and trachealess (trh) but is regulated negatively by forkhead. Scr, the homeotic gene responsible for patterning parasegment 2, is responsible for the activation of every salivary gene that has been tested. As expected, eyg is not expressed in the salivary primordium of Scr-mutant embryos. The trh gene product is necessary for invagination of all salivary duct cells and it is required for expression of downstream duct markers. Because eyg is also expressed in part of the salivary duct primordium, the relationship between trh and eyg was tested in the pathway for duct determination. In wild-type embryos, both trh and eyg expression in the salivary primordium begin early during stage 10. At this stage, eyg expression in trh-mutant embryos is indistinguishable from expression in wild-type embryos. Therefore, initiation of eyg expression in the salivary primordium is independent of trh. In early stage 12, however, eyg expression becomes dependent on trh. Although eyg is expressed strongly in the posterior preduct cells of wild-type embryos, this expression is completely absent in trh-mutant embryos. Therefore it is eyg maintenance, and not its initiation, that depends on trh. Whether trh expression depends on eyg was also tested and it was found that trh expression is unaffected in eyg null-mutant embryos (Jones, 1998).
forkhead plays an important role in establishing the pregland/preduct border by dorsally limiting duct-specific gene expression. trh, like eyg, is also initially expressed throughout the gland primordium. In fkh-mutant embryos, trh transcript never disappears from the pregland cells (Isaac, 1996). Does fkh play a similar negative regulatory role in eyg transcription? When the wild-type eyg expression pattern is compared to that of fkh-mutant embryos, it becomes clear that fkh indeed negatively regulates eyg. eyg expression persists in gland precursors in fkh-mutant embryos. Thus, fkh represses expression of trh and eyg, both of whose expression disappears from the pregland cells at approximately the same time. eyg plays no role in the regulation of fkh expression (Jones, 1998).
Armed with the knowledge that (1) fkh is responsible for the exclusion of both trh and eyg from the pregland cells and (2) trh is necessary for maintenance of eyg expression in the duct cells, it is possible to ask whether fkh represses eyg in the pregland cells simply by repressing trh or if fkh downregulates trh and eyg independently. To address this question, embryos were generated that were doubly mutant for trh and fkh. If the reason for eyg disappearance from the pregland cells in wild-type embryos is disappearance of trh, then it would be predicted that eyg expression would not persist in trh;fkh-mutant embryos. eyg expression, however, does persist in pregland cells in trh;fkh-mutant embryos, suggesting that trh plays no role in eyg repression by fkh. Thus, after
the initial establishment of the salivary primordium by Sex combs reduced, forkhead excludes eyegone expression from the pregland cells so that eyegone's salivary
expression is restricted to the posterior preduct cells. trachealess, in contrast,
activates eyegone expression in the posterior preduct cells (Jones, 1998).
Localized expression of eyg/toe in the thorax is achieved by the activity of two antagonistic factors: the promoting
activity induced by the Iro and pnr genes, and the repressing
activities exerted by the Hh and the Dpp pathways. The latter are probably
mediated by Hh and Dpp target genes that are yet to be identified (Aldaz, 2003).
Both Iro genes and pnr act as activators of eyg/toe
expression. In Iro gene-mutant clones eyg is abolished, and ectopic
Iro gene activity results in ectopic eyg expression. Although
pnr- clones do not lose eyg activity, the
probable explanation is that they show Iro gene activity, which
upregulates eyg. However, ectopic pnr expression induces
eyg activity. Because the Iro gene and pnr expression domains cover the entire
notum, in the absence of any other regulation they would induce eyg
activity in the whole structure (Aldaz, 2003).
The elimination of eyg activity from the scutellum and lateral
notum is caused by the Hh and Dpp pathways. Because the AP compartment border
is displaced posteriorly in the notum, these two pathways are active at high
levels in the posterior region of the mesothorax. Assuming that the two
signals behave as in the wing, Hh activity will be higher in the region close to the AP
border, whereas the effect of Dpp will extend further anteriorly. Thus the
repressive role of Hh will be greater in the proximity of the AP border and
that of Dpp will be greater in more anterior positions. This is precisely what the results indicate. In the region close to the AP border the high Hh levels alone are sufficient to repress eyg. However. in more
anterior positions, close to the border of the eyg domain, Hh levels
are lower and, although Hh is still necessary, it is not sufficient to repress eyg. Here there is an additional requirement for Dpp activity (Aldaz, 2003).
Thus, the part of the notum that does not express eyg can be
subdivided into two distinct zones according to the mode of eyg
regulation: a region close to the AP border that requires only Hh, and a more
anterior region that requires both Hh and Dpp. In the posterior compartment,
the repression of eyg has to be achieved by a different mechanism
because neither the inactivation of the Hh pathway in smo-
clones, nor the inactivation of the Dpp pathway, induces ectopic
eyg activity. A probable possibility is that en itself may
act as repressor (Aldaz, 2003).
The result of the antagonistic activities of the Iro genes and pnr
in one case and of the Hh and Dpp signalling pathways in the other,
subdivides the notum into an eyg/toe expressing domain and a
non-expressing domain. The localized expression of eyg/toe
contributes to the morphological diversity of the thorax, as it distinguishes
between an anterior-central region and a posterior-lateral one. It provides
another example of a genetic subdivision of the body that is not based on
lineage. It also provides an example of a patterning gene acting downstream of the combinatorial code of selector and selector-like genes. Its mode of action supports a model in which the genetic specification of complex patterns, such as the notum, is achieved by a stepwise process involving the activation of a cascade of regulatory genes (Aldaz, 2003).
Control of growth determines the size and shape of organs. Localized signals known as 'organizers' and members of the Pax family of proto-oncogenes are both elements in this control. Pax proteins have a conserved DNA-binding paired domain, which is presumed to be essential for their oncogenic activity. Evidence is presented that the organizing signal Notch does not promote growth in eyes of D. melanogaster through either Eyeless (Ey) or Twin of eyeless (Toy), the two Pax6 transcription factors. Instead, it acts through Eyegone (Eyg), which has a truncated paired domain, consisting of only the C-terminal subregion. In humans and mice, the sole PAX6 gene produces the isoform PAX6(5a) by alternative splicing; like Eyegone, this isoform binds DNA though the C terminus of the paired domain. Overexpression of human PAX6(5a) induces strong overgrowth in vivo, whereas the canonical PAX6 variant hardly effects growth. These results show that growth and eye specification are subject to independent control and explain hyperplasia in a new way (Dominguez, 2004).
Notch (N) signal is activated at the dorsoventral (DV) border of the
Drosophila eye disc and is important for growth of the eye disc. In
this study, the Pax protein Eyg is shown to be a major effector mediating
the growth promotion function of N. eyg transcription is induced by N
signaling occurring at the DV border. Like N, eyg controls
growth of the eye disc. Loss of N signaling can be compensated by
overexpressing eyg, whereas loss of the downstream eyg
blocks the function of N signaling. In addition, N
and eyg can induce expression of upd, which encodes the
ligand for the Jak/STAT pathway and acts over long distance to promote cell
proliferation. Loss of eyg or N can be compensated by
overexpressing upd. These results suggest that upd is a
major effector mediating the function of eyg and N. The
functional link from N to eyg to upd explains how
the localized Notch activation can achieve global growth control (Chao, 2004).
Notch is activated at the DV boundary of the early eye
disc. This equatorial N signal then activates eyg expression at the
transcriptional level. When N signal is reduced, eyg expression is
reduced. When N signal is elevated, eyg expression is induced. Induction of
eyg expression occurs at the DV border between the dorsal
Dl-expressing and the ventral Ser-expressing cells. Furthermore, when the upstream N signal is blocked, overexpression of eyg can rescue the
growth defect in the eye, whereas increasing N signaling cannot rescue the eye-growth defect caused by the downstream eyg gene. This analysis
shows that the induction of eyg by N is dependent on the
ligands Dl and Ser, and involves the effector Su(H)
and the antagonist Hairless. Thus, the localized activation of N signal is
transmitted to the induction of a transcription factor, Eyg, which then
promotes cell proliferation. The Chao study confirms the work of Dominguez (2004), who showed that Notch promotes growth in eyes by acting through Eyegone (Chao, 2004).
Eyg is a transcription factor, so must activate the transcription of some
genes that promote cell proliferation. Upd is reported to act through the
Jak/STAT signaling pathway to promote cell proliferation. upd expression is dependent on eyg and N signaling. Furthermore, when
the upstream N signaling or eyg is reduced, overexpression of
upd can rescue the growth defect. The overgrowth effect
due to overexpression of the upstream N or eyg is blocked
when the downstream upd is defective. The results suggest that upd is a major effector for the growth promotion by N and eyg (Chao, 2004).
These results have demonstrated the functional link from Notch to
eyg to upd in the promotion of eye growth. The link to
upd solved a long-standing problem. N signaling is activated locally
at the border between the dorsal Dl-expressing cells and the ventral
Ser-expressing cells. How does a localized activation of N signal promote cell
proliferation throughout the entire eye disc? The finding of eyg as
the major mediator of N function did not solve the problem, since Eyg is a
transcriptional factor and is expected to affect target gene expression
autonomously. The link from eyg to upd provided a solution,
because Upd is a diffusible signaling molecule. Upd protein can distribute over a
long distance and exert long-range non-autonomous effect to promote cell
proliferation (Tsai, 2004). So the localized N activation can locally activate
eyg, which then turns on upd expression, probably through a
short-range signal. The Upd signal then acts over a long range to promote cell
proliferation in the early eye disc (Chao, 2004).
Although N activates eyg, and
eyg activates upd, these transcriptional activation may be
direct or indirect. When novel DV borders were created by ectopic expressing
Dl or Ser, eyg is induced non-autonomously at the border of
these clones. It is
also noted that in Su(H) mutant clones, mutant cells at the border of
the clone can still express eyg-lacZ. These observations
suggest that N may induce a short-range signal, which then activates
eyg expression. Alternatively, the apparent non-autonomous induction
may be due to perdurance of the reporter protein in cells that were once close
to the clone border. The induction of upd by eyg also may be
indirect. Clonal expression of eyg also induced upd
expression non-autonomously. In addition, based on RNA in situ hybridization,
eyg expression in the eye disc does not extend to the posterior margin, so does not overlap with the expression domain of upd
(Tsai, 2004). These observations suggested that the effect of eyg on upd expression may be indirect. However, an eyg enhancer trap line showed reporter expression extending to the posterior margin
(Dominguez, 2004). Thus, the possibility that Eyg can directly activate the
expression of upd cannot be excluded (Chao, 2004).
The activation of eyg and upd are context dependent.
Nact does not induce eyg expression in antenna
and wing discs. In the eye disc, Nact induces eyg
expression only in the region anterior to the MF, and not within the
wg expression domain in the lateral margin. Similarly,
Nact and eyg can only induce upd
expression at the margin,
but not in the center of the eye disc. Nact induces
upd at the posterior margin but not lateral margins, while
eyg can induce upd in the lateral margins but not in the
posterior margin. The context dependence indicates that additional factors are
involved to determine the specificity of induction (Chao, 2004).
In a late third instar eye disc, eyg is expressed in an equatorial
domain that does not overlap with the disc margin, so eyg cannot induce
upd. In early eye disc, eyg expression domain comes closer
to the posterior margin. Thus, the induction of upd by eyg is
likely at second instar, which is consistent with the timing of upd
expression (Chao, 2004).
Although eyg plays an important role in mediating the
growth-promoting N signal, it is probably not the only effector. In the
eygM3-12 null mutant, ey>Nact does
not rescue the endogenous eye field, but can still induce proliferation to
provide the antennal disc and an extra eye field. Thus, N can induce
proliferation by an eyg-independent mechanism. The effect on antenna
and on eye seems to be separate, because ey>Nact can
induce a large antenna disc with duplicate or triplicate antennal field
without rescue of the eye disc. Because N can induce upd, but not eyg, in the posterior margin, the induction of upd can also be through an
eyg-independent mechanism (Chao, 2004).
Nact can induce overgrowth in the central domain of the
eye disc. In this case, eyg, but not upd, is induced. In addition, the overgrowth does not extend much beyond the clone. Ectopic eyg in the central
domain also induces proliferation without inducing upd. In
eyg1 mutant, there is no upd-lacZ expression in
eye disc, but the eye is only slightly reduced. These results suggest that the N signaling and eyg can induce local proliferation independent of upd (Chao, 2004).
In salivary ducts, breathless (btl) but not Serrate is activated by eyegone. btl codes for a Drosophila FGF receptor homolog that is critical to tracheal and midline glial cell development, while Ser encodes a Notch ligand containing a single EGF repeat. Although neither is required for duct morphogenesis, they are expressed during most of duct development and therefore serve as useful duct markers. Ser and btl expression have been shown to depend on trh (Kuo, 1996). Since trh also regulates eyg, whether eyg might act as an intermediate in the regulation of btl and Ser by trh was also tested. btl expression in duct cells is strongly reduced in embryos lacking eyg. In contrast, expression of Ser is not downregulated in the duct cells of eyg-mutant embryos even though the duct primordia are not fused as in wild-type embryos (Jones, 1998).
In Drosophila, the morphological diversity is generated by the
activation of different sets of active developmental regulatory genes in the
different body subdomains. This study investigates the role of the
homothorax/extradenticle (hth/exd) gene pair in the
elaboration of the pattern of the anterior mesothorax (notum). These two genes
are active in the same regions and behave as a single Hox independent functional unit. Their original uniform expression in the notum is downregulated during development and becomes restricted to two distinct, alpha and ß subdomains. This modulation appears to be important for the formation of distinct patterns in the two subdomains. The regulation of hth/exd expression is achieved by the combined repressing functions of the Pax gene eyegone (eyg) and of the Dpp pathway. hth/exd is repressed in the body regions where eyg is active and that also contain high levels of Dpp activity. Evidence is presented for a molecular interaction between the Hth and the Eyg proteins that may be important for the patterning of the alpha subdomain (Aldaz, 2005).
This study deals with a novel hth/exd function: its patterning role
in the notum. It is not related to the specification of notum identity because
notum identity is not affected by alterations of hth/exd activity. For example, in the absence of hth/exd, the cells still differentiate as notum, if an abnormal one. Conversely, high and uniform Hth levels also produce notum tissue but with abnormal pattern. This
function is only required in part of the notum and is therefore linked to the
modulation of hth expression during the development of the disc. The
final result of this modulation is the appearance of the alpha and ß
subdomains of hth that is reported in this study. These two subdomains
differentiate distinct notum patterns, suggesting that Hth/Exd interact with
other localised products to generate these patterns (Aldaz, 2005).
Thus, there are two principal aspects in the patterning function of
hth/exd: (1) the spatial regulation, that eventually results in the
restriction of its expression to the alpha and ß subdomains, and (2)
the local interactions of Hth/Exd with other products in either of the
subdomains (Aldaz, 2005).
Although hth and exd form a single functional unit, their
mode of regulation is different: exd is expressed ubiquitously but is
regulated at the subcellular level by hth, which promotes Exd nuclear
transport. Therefore, the key element of hth/exd regulation is the
transcriptional control of hth (Aldaz, 2005).
Originally, hth is expressed in all the notum cells and later becomes restricted to the alpha and ß subdomains. Consequently, the principal aspect of hth regulation is the mechanism(s) leading to its repression in the regions outside the alpha and ß subdomains. Two negative regulators have been identified, the
eyg gene and the Dpp pathway, which probably acts through some
unidentified downstream gene. In the notum hth behaves as a
downstream target of both the Dpp pathway and eyg (Aldaz, 2005).
The role of Eyg as a negative regulator of hth is based on the
following observations: (1) the beginning of the modulation of hth
expression in the notum at the early third instar coincides with the
initiation of eyg expression; (2) in eyg mutants the hth domain is expanded, extending to most of the notum; (3) mutant eyg clones show hth derepression in the inter-subdomains region, and conversely, ectopic eyg activity in the ß subdomain represses hth. The fact that this ectopic activity fails to affect hth in the alpha subdomain was expected since eyg and hth are normally co-expressed in this subdomain. In conclusion, eyg suppresses hth in the inter-subdomains region and also acts as a barrier for hth in the eyg/ß-hth
border (Aldaz, 2005).
The role of the Dpp pathway as a negative regulator of hth is
based on results showing that Mad - mutant clones in the
inter-subdomains region show activation of hth. This is in contrast
to the behaviour of those clones in the alpha subdomain, where they have no
effect, or in the ß subdomain, where they show suppression of hth. It is believed that the reason for the latter effect is that eyg is up regulated in those clones, and in turn Eyg suppresses hth. The lack of effect of Mad - clones in the alpha subdomain is probably due to the low activity of Dpp in that region. In principle, the observation that the high activity levels generated in the TkvQD clones suppress hth in this subdomain supports this view. Expectedly, TkvQD clones do not affect hth expression in the ß subdomain, because it normally possesses high Dpp activity levels (Aldaz, 2005).
Taking all the results together, the following model of
hth regulation is proposed. Since hth is originally expressed in all trunk embryonic cells and in all the notum cells in the early disc, the
regulation of hth during wing disc development essentially reflects
local repression in specific parts of the disc. The basic idea is that
hth is repressed by the joint contribution of eyg and
high/moderate levels of the Dpp pathway. Neither of these elements can repress
hth individually. Although eyg appears to act uniformly in
its domain, the repressing activity of Dpp is concentration dependent. Within
the eyg domain, the hth alpha subdomain is located in the
anterior region, in which the Dpp levels are too low to be effective and Eyg
alone cannot repress hth/exd. In the inter-subdomains region the Dpp
levels are high enough to repress hth, since here it acts together with
Eyg. The ß subdomain is outside the eyg domain and therefore in
the absence of Eyg even the high Dpp levels are not capable of repressing
hth/exd. The model is also supported by the experiments of
overexpressing eyg. The eyg-expressing clones in the ß
subdomain suppress hth because the two repressors are active in the
clones, while they have no effect in the alpha subdomain because it normally
contains high eyg levels. In principle the experiments overexpressing
the Dpp pathway (TkvQD clones) appear to support the model. These
clones have no effect in the ß subdomain, which normally possesses high
Dpp activity levels, but they suppress hth in the alpha subdomain.
However, these clones are known to suppress eyg and
therefore hth should not be repressed according to this model. It is
possible that in certain circumstances the very high Dpp activity levels
induced by these clones may be sufficient to down regulate hth, even
in the absence of eyg (Aldaz, 2005).
The presence of two distinct repressors may suggest that the hth
promoter region contains binding sites for Eyg and for Mad/Medea
that would be responsible for the transcriptional repression. The ubiquitous
expression in the absence of these two repressors may be due to a constitutive
promoter (Aldaz, 2005).
The second aspect of the late patterning function of hth/exd
arises from the observation that the alpha and ß subdomains form
different patterns with similar levels of hth. This suggests the
existence of interactions between Hth/Exd and products specifically localised
to the different subdomains. In the case of the alpha subdomain, the obvious
candidate for the interaction is Eyg. The joint activity of
hth/exd and eyg specifies a notum pattern that is different
from those specified by each of these genes alone (Aldaz, 2005).
The finding that the Eyg and Hth proteins associate to form a complex in
vitro suggests a mechanism to achieve the pattern difference between the
alpha and the ß subdomains. As it has been shown to be the case for the
in vivo specificity of the Hox genes, the association of Hth/Exd with the
different Hox products results in higher affinity and specificity for target
sites. Here, the formation of an Eyg/Hth/Exd complex in the alpha subdomain may result in a constellation of gene activity different from that in the ß subdomain where Eyg is not present. In the latter subdomain hth/exd may act alone, for after all the two genes encode transcription factors. Alternatively, the Hth/Exd products may interact with some other yet
unidentified co-factor (Aldaz, 2005).
An interesting aspect of the interaction of hth/exd and
eyg is that it acts in two different ways. At the gene regulation
level, eyg participates in the spatial control of hth/exd
activity, but where the two genes are co-expressed their proteins interact,
presumably to contribute to the in vivo affinity and specificity for target
genes (Aldaz, 2005).
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