pannier
pnr is localized to the dorsal 30% of the blastoderm embryo. This is the region that gives rise to amnioserosa [Images] and dorsal epidermis. Zerknüllt and Decapentaplegic are also localized to the dorsal epidermis in a broader domain. Unlike ZEN and DPP, PNR is restricted to 20-60% (anterior to posterior) of the egg length. Expression is seen in five stripes, reflecting parasegmental units. The posterior boundary expands to the proctodeum during the early phase of germ band extension [Images]. Throughout Dorsal closure, pnr declines in expression and is confined to a band of single cells on either side of the dorsal midline (Winick, 1993).
The Drosophila lymph gland is a hematopoietic organ and, together with prospective vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes), arises from the cardiogenic mesoderm. Clonal analysis provided evidence for a hemangioblast that can give rise to two daughter cells: one that differentiates into heart or aorta and another that differentiates into blood. In addition, the GATA factor gene pannier (pnr) and the homeobox gene tinman (tin), which are controlled by the convergence of Decapentaplegic (Dpp), fibroblast growth factor (FGF), Wingless (Wg) and Notch signaling, are required for the development of all cardiogenic mesoderm, including the lymph gland. An essential genetic switch differentiates between the blood or nephrocyte and vascular lineages involves the Notch pathway. Further specification occurs through specific expression of the GATA factor Serpent (Srp) in the lymph-gland primordium. These findings suggest that there is a close parallel between the molecular mechanisms functioning in the Drosophila cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm (Mandal, 2004).
Blood and vascular cells in the vertebrate embryo are thought to derive from oligopotent progenitor cells, called hemangioblasts, that arise in the yolk sac and in the aorta-gonadal-mesonephros (AGM) mesenchyme. A close relationship between blood and vascular progenitors is well established, but in vivo evidence that a single cell can divide to produce a blood cell and an endothelial cell is lacking in vertebrate systems. Similarly, the molecular mechanism that distinguishes between the two lineages is not well understood. To address these issues in a simple, genetically amenable system, the genetic control of hematopoiesis was analyzed in Drosophila. The results show that there are close lineage relationships between hematopoietic and vascular cells, similar to those present in the AGM of mammalian systems. Evidence is provided for conserved cassettes of transcription factors and signaling cascades that limit the pool of hemangioblastic cells and promote the blood versus vascular fate (Mandal, 2004).
In the mature Drosophila embryo, the lymph gland is formed by a paired cluster of ~20 cells flanking the aorta. The aorta and heart represent a contractile tube lined by a layer of myoepithelial vascular cells called cardioblasts. The cells flanking the aorta and heart posterior to the lymph gland are the pericardial cells, which function as excretory cells (nephrocytes). Lymph gland progenitors express the prohemocyte marker Srp and ultrastructurally resemble prohemocytes that develop at an earlier stage from the head mesoderm. Monitoring expression of the zinc-finger protein Odd-skipped (Odd) shows that the lymph gland originates from the dorsal thoracic mesoderm. Odd is expressed in segmental clusters in the dorsal mesoderm of segments T1-A6. The three thoracic Odd-positive clusters coalesce to form the lymph gland, whereas the abdominal clusters formed the pericardial nephrocytes (Mandal, 2004).
Lymph-gland progenitors, cardioblasts and pericardial cells are closely related by lineage. Labeled 'flipout' (FLP/FRT) clones were induced in embryos aged 3-4 h such that the clones contained only 2-4 cells. Of the two-cell clones, ~50% contained cardioblast and lymph-gland cells; the other clones comprised either cardioblasts or lymph-gland cells alone. Mixed clones were recovered at the late third larval stage. The finding of mixed clones indicates that the cardiogenic mesoderm of D. melanogaster contains oligopotent progenitors that, up to the final division, can give rise both to Srp-positive blood-cell progenitors that form the lymph gland and to vascular cells (Mandal, 2004).
The cardiogenic mesoderm forms part of the dorsal mesoderm, which requires the homeobox protein Tin and the GATA factor Pnr. In embryos with mutations in tin or pnr, the lymph gland was absent. Maintenance of Tin expression in the dorsal mesoderm requires the activity of at least two signaling pathways regulated by Dpp (the Drosophila homolog of transforming growth factor-ß) and Heartless (Htl; one of the D. melanogaster homologs of the FGF receptor); the dependence of cardioblast and pericardial nephrocyte development on these signaling pathways has been documented. Lymph-gland progenitors did not develop in loss-of-function dpp and htl mutants (Mandal, 2004).
Between 6 h and 8 h of development, the dorsal mesoderm splits into the cardiogenic mesoderm and the visceral mesoderm. The cardiogenic mesoderm is regulated positively by Wg and negatively by Notch. Lack of Wg signaling results in the absence of all cardiogenic lineages including lymph gland. Notch signaling has the opposite effect and restricts cardiogenic mesodermal fate. Notch is active in the dorsal mesoderm from 6 h to 10 h of development. Eliminating Notch during the first half of this interval by raising embryos homozygous with respect to the temperature-sensitive allele Nts1 at the restrictive temperature resulted in substantially more cardioblasts, pericardial cells and lymph-gland progenitors (Mandal, 2004).
Lymph-gland progenitors, cardioblasts and pericardial nephrocytes are specified in the cardiogenic mesoderm around the phase of germband retraction 8-10 h after fertilization. At this stage, Tin, which was initially expressed in the whole cardiogenic mesoderm, becomes restricted to a narrow medial compartment containing the cardioblasts. Pnr follows the same restriction. Cells located at a more lateral level in the cardiogenic mesoderm give rise to lymph-gland progenitors (in the thoracic domain) and pericardial nephrocytes (in the abdominal domain) and activate the gene odd. Slightly later, Srp is expressed in lymph-gland progenitors. As reported for the early hemocytes derived from the embryonic head, srp is centrally involved in lymph-gland specification. In srp-null embryos, Odd-expressing cells still formed a lymph gland-shaped cluster flanking the aorta, but these cells also express the pericardial marker Pericardin (Prc), suggesting that they lose some aspects of hemocyte precursor identity or gain properties of nephrocytes. As a countercorrelate, ectopic expression of Srp in the whole cardiogenic mesoderm directed by mef2-Gal4 induces pericardial cells to adopt lymph-gland fate (Mandal, 2004).
Downregulation of tin and pnr in cells in the lateral domain of the cardiogenic mesoderm is essential for lymph-gland specification. Ectopic expression of tin or pnr by twist-Gal4 (or mef2-Gal4) causes a marked reduction in the number of lymph-gland and pericardial cells. The antagonistic effect of tin on lymph-gland progenitors resembles its earlier role in the head mesoderm that gives rise to the larval blood cells; here too, ectopic expression of tin causes a reduction in the number of hemocytes (Mandal, 2004).
Inhibiting tin and upregulating odd and srp requires input from the Notch signaling pathway. A function of Notch at 6-8 h in specification of the cardiogenic mesoderm is described. Reducing Notch function between 8 h and 10 h causes an increase in the number of cardioblasts and a concomitant loss of pericardial and lymph-gland cells. Overexpressing an activated Notch construct causes a marked increase in lymph-gland size. This late requirement for Notch signaling is separable from the earlier role of Notch in restricting the overall size of the cardiogenic mesoderm. Thus, the sum total of cardioblasts and pericardial or lymph-gland cells in Nts1 embryos shifts between 8 h and 10 h and does not differ substantially from that in wild type, whereas a combined effect on cell number and cell fate is seen in embryos with a Notch deletion. In these embryos, the cardiogenic mesoderm is hyperplasic and develops as cardioblasts at the expense of lymph-gland progenitors and pericardial nephrocytes. The dual role of Notch in restricting the numbers of a pluripotent progenitor pool and in distinguishing between the progeny of these progenitors is reminiscent of the function of Notch in sense-organ development (Mandal, 2004).
Lymph-gland formation is restricted to the thoracic region by positional cues that are provided by expression of the homeobox proteins of the Antennapedia and Bithorax complex. Specifically, Ultrabithorax (Ubx), which is expressed in segments A2-A5 of the cardiogenic mesoderm, inhibits lymph-gland formation. Loss of Ubx results in the expansion of the lymph-gland fate into the abdominal segments. Conversely, overexpression of Ubx driven by mef2-Gal4 causes the transformation of lymph-gland progenitors into pericardial nephrocytes (Mandal, 2004).
These findings are suggestive of a model of lymph-gland development in Drosophila that is similar to mammalian hematopoiesis. Lymph-gland progenitors develop as part of the cardiogenic mesoderm that also gives rise to the vascular cells (aorta and heart) and to excretory cells. Similarly, progenitor cells of the blood, aorta and excretory system are closely related both molecularly and developmentally in mammals, where they form part of the AGM. Specification of the cardiogenic mesoderm requires the input of FGF and Wg signaling, as in vertebrate hematopoiesis, where the AGM region is induced in response to several converging signaling pathways including FGF, BMP and Wnt (Mandal, 2004).
The cardiogenic mesoderm in Drosophila evolves from the dorsal mesoderm and requires input from the Htl, Dpp, Wg and Notch (N) signaling pathways. The cardiogenic mesoderm then differentiates into lymph gland, vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes). A subpopulation of cardioblasts and lymph-gland cells is derived from one progenitor (hemangioblast; HB). Essential for the differentiation of the cardiogenic mesoderm is the Notch-Delta (Dl)-dependent restriction of Tin and Pnr to cardioblasts and the expression of Srp in the lymph gland. In vertebrates, similar cell types are derived from a mesodermal domain called the AGM, which also requires the input of FGF, BMP and Wnt signaling. A subset of AGM-derived cells has been proposed to constitute hemangioblasts, which produce blood progenitors and endothelial cells (Mandal, 2004).
These findings show that in Drosophila, the cardiovascular and blood-cell lineages are differentiated by an antagonistic relationship between Tin or Pnr expression in the cardioblasts and Srp expression in the lymph-gland progenitors. In vertebrates, GATA factors also have a pivotal role in specifying different lineages among blood-cell progenitors, although not much is known about what differentiates between blood progenitors as a group and endothelial progenitors. The results indicate that this step is driven by input from the Notch signaling pathway. In the thoracic cardiogenic mesoderm, Notch antagonizes tin and pnr expression and aortic cardioblast formation, and promotes srp expression and the development of lymph-gland progenitors. In vertebrates, Notch signaling is also involved in both blood and vascular development. The role of Notch during AGM morphogenesis remains to be investigated (Mandal, 2004).
Cardioblasts and lymph-gland cells can arise from the division of a single cardiogenic mesodermal cell, which should be called a hemangioblast. A previous study induced clones in the cardiogenic mesoderm but used only Tin as a marker. This study also yielded mixed two-cell clones comprising a cardioblast and a nonlabeled cell, which, in light of the current findings, must be interpreted as a lymph-gland cell. Hemangioblasts have been proposed in vertebrates, although the definitive experiment in which a precursor is marked and its lineage is tracked has not been done. Blast colony-forming cells that give rise to both lineages in vitro and common markers that belong to both cell types in vivo have been identified, but direct evidence for the existence of a common precursor has not yet been found. This study, using genetic analysis of two-cell clones, establishes the existence of such a population in Drosophila. On the basis of these results, and given the conservation of the signaling and transcriptional components described here, the prediction is that many cells of the AGM in vertebrates may give rise to only blood or only vascular cells, but a number of intermixed hemangioblasts may give rise to mixed lineages. Future genetic screens aimed at finding components in early lymph-gland development will probably identify additional pathways and strategies important for vertebrate hematopoiesis (Mandal, 2004).
pannier is expressed in a dorsal stripe in the adult thorax. In the thoracic disk pannier is expressed in a broad dorsal area, and in the dorsal half of the eye-antennal disc (Hietzler, 1996).
The Bar homeobox genes function as latitudinal prepattern genes in the developing
Drosophila notum. In Drosophila notum, the expression of achaete-scute
proneural genes and bristle formation have been shown to
be regulated by putative prepattern genes expressed
longitudinally. The two Bar locus genes may belong to a different
class of prepattern genes expressed latitudinally: it is
suggested that the developing notum consists of checker-square-
like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate
the formation of microchaetae within the region of
BarH1/BarH2 expression through activating achaete-scute.
Presutural macrochaetae formation also requires Bar
gene activity. Bar gene expression is restricted in dorsal and posterior regions by Decapentaplegic
signaling, while the ventral limit of the expression domain
of Bar genes is determined by wingless, whose
expression is under the control of Decapentaplegic signaling (Sato, 1999).
The Drosophila notum is considered genetically divided into
several longitudinal, side by side, domains whose boundaries are determined by pannier, wingless and iroquois expression (listed respectively from medial to lateral). To further
clarify relative locations of pnr, wg and iro expression areas,
third-instar larval and pupal future notum were stained with
various combinations of molecular markers. In larval and
pupal future notum, pnr-Gal4 is expressed medially and iro-lacZ
laterally. pnr-Gal4 and iro-lacZ
domains partially overlap one another, and wg-lacZ (or Wg) expression is
noted in the pnr-iro overlapping region and its immediate
neighbors. Bar homeobox genes may belong to an additional class of notal
subdivision genes. Staining for BarH1 indicates that BarH1 is expressed latitudinally (anterior vs. posterior) in the anterior-most region of future notum and postnotum. BarH1 expression begins at early to mid third instar. Anti-Ac antibody staining and neur-lacZ expression
indicates PS macrochaetae are situated in the vicinity of
posterior-ventral corners of the anterior BarH1 expression
domain. BarH1 and BarH2 are referred to as Bar collectively and the anterior portion of
the prescutum or its precursor expressing Bar is referred to as Bar
prescutum. The Bar expression domain overlaps that of pnr, wg and iro. Bar expression similar to that in wing discs is observed in haltere discs (Sato, 1999).
It is concluded that a checker-board-like subdivision of future notum is regulated by
putative prepattern gene expression.
Future notum may be divided into square subdomains in a
checker-board-like manner, each with its own unique
combinations of prepattern gene expression.
Putative prepattern genes, iro and pnr, form longitudinal
domains. Bar homeobox genes form
the anterior-most domain. This is
the first demonstration of the presence of latitudinal, front to back, prepattern
genes in the notum. Bristle formation in each subdomain may
be positively regulated by a region-specific combination of
prepattern genes. Consistent with this, microchaetae formation in the anterolateral
prescutum (the lateral Bar prescutum), where Bar and iro are
coexpressed, requires the concerted action of Bar
and iro (Sato, 1999).
In mutants, cells of the amnioserosa and part of the dorsal epidermis die. This causes a lethal hole in the dorsal cuticle of the larva (Ramain, 1993).
A genetic and phenotypic analysis of the gene pannier is described. Animals mutant for strong alleles
die as embryos in which the cells of the amnioserosa are prematurely lost. This leads to a dorsal
cuticular hole. The dorsal-most cells of the imagos are also affected: viable mutants exhibit a cleft
along the dorsal midline. Pannier mRNA accumulates specifically in the dorsal-most regions of the
embryo and the imaginal discs. Viable mutants and mutant combinations also affect the thoracic and
head bristle patterns in a complex fashion. Only those bristles within the area of expression of pannier
are affected. A large number of alleles have been studied; they reveal that pannier may have opposing
effects on the expression of achaete and scute leading to a loss or a gain of bristles. Certain alleles of pannier are sensitive to the dose of extra-machrochaetae. In those parts of the epithelium where both are present, it is possible that pnr and emc function together in the same ac-sc repression pathway (Heitzler, 1996).
The regulation of cardiac gene expression by GATA zinc
finger transcription factors is well documented in
vertebrates. However, genetic studies in mice have failed to
demonstrate a function for these proteins in cardiomyocyte
specification. In Drosophila, the existence of a cardiogenic
GATA factor has been implicated through the analysis of a
cardial cell enhancer of the muscle differentiation gene Mef2.
The GATA gene pannier is expressed in
the dorsal mesoderm and required for cardial cell
formation while repressing a pericardial cell fate. Ectopic
expression of Pannier results in cardial cell overproduction,
while co-expression of Pannier and the homeodomain
protein Tinman synergistically activate cardiac gene
expression and induce cardial cells. The related GATA4
protein of mice likewise functions as a cardiogenic factor
in Drosophila, demonstrating an evolutionarily conserved
function between Pannier and GATA4 in heart
development (Gajewski, 1999).
tinman gene function is required for heart development in
Drosophila. The
initial programming of the cardiac lineage occurs at a time
when tin is broadly expressed in the dorsal mesoderm. A subset of the tin-expressing cells will become heart
precursors, appearing in 11 clusters along the dorsalmost part
of the mesoderm. The Mef2 enhancer-lacZ fusion
gene marks heart progenitors at stage 11 and will eventually
be expressed in four pairs of cardial cells per segment of the
dorsal vessel. Thus, the tin expression domain is significantly larger
than the territory of heart precursor specification, suggesting
the involvement of additional factors in the formation of these
cells.
The Mef2 heart enhancer requires the presence of at least
three elements for its activity, including two Tin binding sites
and one GATA sequence. The GATA gene pnr is expressed in cells
of the dorsal ectoderm around the time of heart cell
specification. However, there is no report of pnr transcription in the
mesoderm. To investigate this possibility, embryos were stained
for PNR mRNA and embryo cross-sections were examined. At late
stage 10, gene expression is observed in the dorsal ectoderm
of the germband-extended embryo. PNR mRNA was detected in four clusters of cells located in the dorsalmost part
of the mesoderm that corresponds to the cardiogenic region.
Additionally, a pnr mesodermal enhancer has been identified that
directs lacZ expression in the heart-forming region, but not in
the overlying ectoderm. Therefore, pnr is
expressed in the cardiogenic mesoderm where it could function
in cardial cell specification and the regulation of Mef2
transcription (Gajewski, 1999).
The Mef2 IIA341 enhancer is active in both cardial and
ventral muscle founder cells and a GATA site is
needed for the cardial cell expression.
To determine if pnr function is important for enhancer activity
in either of these cell types, the expression of
the enhancer-lacZ fusion was examined in pnr mutant embryos. A strong reduction of reporter gene expression is observed in the dorsal
mesoderm of the mutants, while normal lacZ
expression is detected in the ventral muscle precursors. These results suggest Pnr is a transcriptional regulator of
the Mef2 heart enhancer, consistent with the observation that
the protein can bind the essential GATA sequence in an
electrophoretic mobility shift assay.
Alternatively, or in addition, these results could be indicative
of a requirement of pnr function for the formation of the Mef2-expressing cardial cells (Gajewski, 1999).
To determine if pnr function is essential for the
specification of cardial cells, wild-type and mutant embryos
were stained for Mef2 protein that serves as a marker for
these cells. A distinct row of cardial cells is observed in a
lateral view of normal embryos at stage 13 as they migrate
dorsally along the overlying ectoderm during the process of
dorsal vessel formation. In contrast, these cells are
greatly diminished or completely absent from the dorsal
mesoderm of mutant embryos. To assess the
formation of the pericardial cells in the same genetic
background, embryos were stained for Eve protein, which is a
marker for a subset of these cells. Eleven clusters of Eve-positive
cells, each comprising three or four cells, are detected
in wild-type embryos at stage 12. In contrast, an
overabundance of Eve-expressing pericardial cells is observed
in the pnr embryos. These results suggest that pnr
function is vital to the formation of cardial cells, while
simultaneously playing an important role in controlling the
production of at least one pericardial cell type.
Thus, forced mesodermal expression of Pnr leads to an
overproduction of cardial cells and the generation of supernumerary cardial cells occurs at the
expense of Eve pericardial cells (Gajewski, 1999).
Certain NK-2 class homeodomain and GATA family proteins
have been shown to physically interact in their cooperative
activation of gene expression in cell culture systems. To test
the possibility that Tin and GATA factors could functionally
interact in an embryological context, the tin, pnr
and mGATA4 genes were expressed independently or in combination in
Drosophila embryos. When tin, pnr or mGATA4 are
expressed alone in the twi enhancer-expressing cells, the Mef2
heart enhancer is activated ectopically in the cephalic (tin) or dorsal (pnr and mGATA4) mesoderm.
Since Tin is a known regulator of the Mef2 enhancer, it could be
activating the Mef2 sequence in the head region through its
fortuitous interaction with a co-factor normally expressed in
these cells. The results are striking when both Tin and either
of the GATA factors are co-expressed under the control of the
twi-Gal4 driver. A cardial cell marker is now
activated in both the cephalic region and throughout the dorsal
and ventral trunk mesoderm. Likewise, a strong ectopic
expression of the Mef2 heart enhancer is detected in ventral
midline cells of the developing CNS. The data point to a
combinatorial interaction of Tin and the two GATA factors in
the de novo activation of the cardial cell marker in both
mesodermal and non-mesodermal cells. They also suggest
these genetic combinations are inducing a cardial cell fate
along the ventral midline of the CNS (Gajewski, 1999).
In summary, the discovery of early heart phenotypes in pnr
mutant embryos, coupled with the demonstration of uniquely
conserved cardiogenic abilities of Pnr and GATA4, provide
novel evidence for the function of GATA family members in
the specification of a heart cell type. In an embryological
context, these proteins can work with the Tin homeodomain
factor to program cells into an apparent cardial fate in both
mesodermal and non-mesodermal cell types. This genetic
combination appears to be essential, but not necessarily
sufficient, for cellular commitment to the cardiac lineage as
other factors may contribute to the specification process.
Additional studies using the Drosophila cardiogenic assay
should prove instrumental in revealing other key members of
this genetic program (Gajewski, 1999)
The pannier gene encodes a GATA transcription
factor and acts in several developmental processes in
Drosophila, including embryonic dorsal closure,
specification of cardiac cells and bristle determination. pnr is expressed in the mediodorsal parts of
thoracic and abdominal segments of embryos, larvae and
adult flies. Its activity confers cells with specific adhesion
properties that make them immiscible with non-expressing
cells. Thus there are two genetic domains in the dorsal
region of each segment: a medial (MED) region where pnr
is expressed and a lateral (LAT) region where it is not. The
homeobox gene iroquois is expressed in the LAT
region. These regions are not formed by separate
polyclones of cells, but are defined topographically (Calleja, 2000).
The domain of expression of pnr in adult flies is seen directly
in pnr-Gal4/UAS-y flies or in flies or imaginal discs of the
genotype pnr-Gal4/UAS-lacZ. It is restricted to a
dorsal region of the head, thorax and abdomen. In the head,
pnr is expressed in the dorsal region of the eye and in the head
capsule and will not be considered further. In the dorsal mesothorax pnr covers a longitudinal band that occupies about 40% of the notum and extends from the dorsal midline to the medial zone. It is delineated by a straight border that is aligned with, and just lateral to, the dorsocentral bristles. In
the wing disc, pnr is expressed in the region that contains the
progenitor cells of the medial region of the adult notum.
iro is expressed in both the wing and the thorax, but only the thoracic expression is relevant here. In the second larval instar iro is expressed in all prospective thoracic cells but, in third instar discs, it is restricted to the lateral region, indicating that
there is a retraction of the iro thoracic domain during disc
development. The expression of pnr and iro were compared in the
thorax of late third instar discs: they are expressed in distinct
and complementary subdomains that together cover
the entire mesothorax. Their expression extends to both the A
(notum) and the P (postnotum) mesothoracic compartments as
indicated by double staining for pnr and en or iro and en. The domain of wg in the mesothorax overlaps with
that of pnr and iro but is restricted to the A compartment. The expression of pnr, iro, wg and en in the
haltere disc is the same as in the wing disc, indicating that the
two discs share the same genetic organisation.
In the abdominal segments, pnr defines a medial subdomain
that occupies about 35% of each tergite. Unlike the notum, there is no morphological landmark close to the pnr expression boundary and the bristle and pigment patterns are similar on either side (Calleja, 2000).
Ectopic pnr in the wing induces MED thoracic
development, indicating that pnr specifies the identity of the
MED regions. Correspondingly, when pnr is removed from
clones of cells in the MED domain, they sort out and
apparently adopt the LAT fate. It is proposed that (1) the
subdivision into MED and LAT regions is a general feature
of the Drosophila body plan and (2) pnr is the principal gene
responsible for this subdivision. It is argued that pnr acts like
a classical selector gene but differs in that its expression is
not propagated through cell divisions (Calleja, 2000).
The finding that pnr negatively regulates iro
suggests that the restriction of iro expression to the LAT
subdomain, and hence the appearance of two distinct
subdomains, is a result of pnr activity in the MED region. In
cases such as ap-gal4/UAS-pnr flies in which the restricted
activity of pnr is replaced by uniform expression in all
presumptive notal cells, the subdivision of the notum does not
occur, instead only the MED pattern develops. Thus
restriction of pnr expression to the MED region is a
prerequisite for the partitioning of the notum (Calleja, 2000).
In addition to its function in
partitioning, pnr also specifies the pattern of the MED region.
It participates together with selector genes in a 'genetic
address' that determines the
identity of MED regions of the thoracic and abdominal
segments. In the notum, the activity of pnr is necessary for the
development of the characteristic pattern of the MED region,
whereas its absence allows expression of iro and consequently
formation of the LAT pattern. In the abdomen, pnr is also
required for the development of the MED region. The
developmental capacity of pnr and the binary mechanism in
which it participates are clearly demonstrated by the ectopic
expression experiments that show that Pnr induces different
patterns depending on the local genetic context. In the wing
blade, Pnr induces the formation of the MED notum in the
anterior wing and MED postnotum in the posterior wing. This
result suggests that en is contributing together with pnr to the
genetic address of the MED regions. The DV compartmental
segregation is not recognized by pnr; clones of pnr-expressing
cells in the dorsal and ventral wing compartments differentiate
the same MED-like pattern. But this is not so surprising: no
DV segregation takes place within the normal domain of pnr
expression (Calleja, 2000).
In the haltere disc, the response to pnr activity may be
modulated by the function of Ultrabithorax, the selector
gene that discriminates between haltere and wing development. In the abdomen, ectopic pnr
expression in the ventral region (sternites) induces the
development of a dorsal pattern (tergite). Here the response to
pnr function is probably modulated by the corresponding
genes of the BX-C. This
transformation appears to be similar to that described for
ectopic wg activity in the ventral abdomen. It is possible that Pnr may derepress wg or expand the domain of wg in the sternites and pleurae.
Alternatively, the transformations induced by wg may be
mediated by activation of pnr. Other features of pnr are
reminiscent of classical selector genes. For example, the effect
of pnr on cell affinities is a property shared by selector genes
and is used to keep groups of cells from mixing during growth (Calleja, 2000).
Thus the mode of action of pnr is very similar to that of
selector genes like Ubx, en or ap. The only difference between
the segregation of the MED and LAT subdomains and the AP
or the DV compartment segregations is the manner whereby
activity of the genes is maintained. Expression of en or ap is
inherited unchanged by the cellular progeny, but this is not so
in the case of pnr or iro. The really critical
outcome is thought to be the partitioning of groups of cells into distinct
genetic subdomains. The manner in which the genes
responsible for segregating the genetic subdomains, be these
en, ap or pnr, maintain their activity may be of secondary
importance. If pnr expression is not inherited by the cell
progeny, there has to be some other mechanism to maintain
activity in the appropriate region. pnr is likely to respond to
threshold levels of some specific signal(s) which would
probably emanate from either the midline or from the lateral
margin of the dorsal field (Calleja, 2000).
pnr and iro are not the only developmental genes that define
the identity of body regions by a mechanism not based on cell
lineage. In the leg disc, the distinction between proximal and
distal regions results from the genetic interface between Dll
and hth-exd, which determines the development of the
appendage in the proximodistal axis. This genetic border is not based
on lineage and, as in the case of pnr
in the notum, Dll activity confers specific adhesion properties
to cells causing them to sort out from non-expressing cells. The distinction between external and
internal analia is based on the differential activity of Dll within
the domain of the Hox gene caudal, and this too is lineage
independent. Recent work on the
omb domain in the wing indicates that omb activity is not inherited by cells. As in the
case of pnr, it has been shown that Dll and omb can induce
appendage structures if expressed ectopically, suggesting that their
products determine the identity of different body regions. In
the abdomen, the A compartment is subdivided into anterior
and posterior domains. These domains respond to Hedgehog
in very different ways and yet the border between them is not
colinear with a lineage boundary (Calleja, 2000 and references therein).
The process of compartmentation is an epigenetic
mechanism by which groups of cells become geographically
divided into subgroups that acquire characteristic and distinct
genetic identities. Although compartmentation is normally
associated with cell lineage segregations, the results presented here indicate
that such segregations are not the defining feature of the
process. The MED and LAT subdomains that are reported here
are not segregated by cell lineage and yet they possess all other
features associated with compartments: they originate by
subdivision of groups of presumptive cells, are delimited
by sharp boundaries, and are genetically specified by the
combinatorial activity of a set of selector genes. The
subdivision of the leg into distinct genetic domains or that of the
analia may be other examples of genetic partitions without the concourse of lineage segregations. The principal difference with the MED/LAT subdivision is that the latter affects embryonic, larval as well as adult segments and is therefore a basic feature of the body plan (Calleja, 2000).
Multitype zinc-finger proteins of the class Friend of GATA/U-shaped (Ush) are known to function as transcriptional regulators of gene expression
through their modulation of GATA factor activity. To better understand intrinsic properties of these proteins, the
expression and function of the ush gene during Drosophila embryogenesis was investigated. ush is dynamically expressed in the embryo, including
several cell types present within the mesoderm. The gene is active in the cardiogenic mesoderm, and a loss of function results in an
overproduction of both cardial and pericardial cells, indicating a requirement for the gene in the formation of these distinct cardiac cell
types. Conversely, ectopic expression of ush results in a decrease in the number of cardioblasts in the heart and the inhibition of a cardial cell enhancer normally
regulated by the synergistic activity of the Pannier and Tinman cardiogenic factors. These findings suggest that, similar to its known function in thoracic bristle
patterning, Ush functions in the control of heart cell specification through its modulation of Pannier transcriptional activity. ush is also required for mesodermal cell
migration early in embryogenesis, where it shows a genetic interaction with the Heartless fibroblast growth factor receptor gene. Taken together, these results
demonstrate a critical role for the Ush transcriptional regulator in several diverse processes of mesoderm differentiation and heart formation (Fossett, 2000).
The ush gene exhibits a dynamic pattern of expression during
embryogenesis. Gene transcripts are first detected at high levels in
the primordium of the amnioserosa at stage 5. Additional expression is
observed in germ band extending embryos, in cells of the developing
anterior and posterior midgut, and in hemocyte precursors present in the
cephalic mesoderm. By stage 11, ush RNA
is detected in the dorsal ectoderm and in precursor cells of the
hemocytes and fat body. By late embryogenesis, ush expression is greatly diminished, but transcripts are still observed in the dorsal ectoderm during dorsal
closure and cells within, or associated with, the central nervous system. To
investigate the possible expression of ush in mesodermal
cells underlying the dorsal ectoderm, cross sections of
embryos at stage 11 were examined. ush RNA is detected in a changing
pattern in this germ layer, initially throughout most of the mesoderm and then in subpopulations of cells, including precursors of the fat body, visceral mesoderm, and cardiogenic mesoderm. Therefore,
ush is expressed in the dorsal mesoderm, where it could
function in the early stages of heart formation (Fossett, 2000).
pnr mutant embryos show a loss of contractile cardial cells
and an overproduction of certain nonmuscle pericardial cells in the
heart-forming region. To identify a possible role for ush in these cardiogenic processes,
alterations in cardiac cell production were sought in mutant embryos. The
ush alleles used in this analysis were
ush1 and
ush2, believed to be hypomorphic
mutations of the gene, and Df(2L)al, a chromosome deletion
that represents a ush null mutation. The D-mef2
heart enhancer-lacZ fusion gene serves as a cardial cell marker, since it is detected in progenitors of these cells around stage 11 and thereafter in two, then four cardioblasts per hemisegment of the
forming dorsal vessel. Embryos homozygous for either a
point mutation or deletion of the gene show an increase in the number
of cells expressing the reporter gene, as compared with the wild-type
embryo. In
ush1 embryos, a few hemisegments contain up to nine positive cells with an average of six cardial cells present in many clusters. In
ush-deficiency embryos, a comparable increased density of
cardial cells is found (Fossett, 2000).
Mef2 protein also marks cardioblasts; it is detected in the nuclei
of all cardial, but not pericardial cells of the forming dorsal vessel.
In wild-type embryos at stage 13, the germ band has retracted with
cardioblasts migrating dorsally, separating from the dorsal somatic
muscles. A lateral view at this stage shows a single row of cells that
contains six stained nuclei per hemisegment. In
contrast, ush mutant embryos possess supernumerary
cardioblast nuclei. ush1 and ush2 embryos contain up to 12 nuclei per hemisegment with eight cells per cluster observed on average. Similar results have also been obtained with ush-deficiency embryos.
Therefore, reducing or completely eliminating ush function
leads to an increased production of cardial cells. Intriguingly, the
ush heart phenotype uncovered by the analysis of these two
markers directly contrasts the absence of cardial cells observed in
pnr loss-of-function embryos (Fossett, 2000).
Production of pericardial cells was quantitated in
ush mutant embryos, using Eve protein as a marker for a
subset of these cells. In wild-type embryos at stage 12, there exist 11 Eve-positive clusters within the dorsal mesoderm, each containing about
three cells. In contrast, the number of
Eve-expressing pericardial cells increases in homozygous
ush1 and ush2
embryos to an average of 5-6 per cluster. A similar increase in pericardial cell number is also observed in homozygous Df(2L)al embryos.
Thus, ush gene activity is required to prevent the
overproduction of this pericardial cell type, a function that has also
been ascribed to the pnr gene (Fossett, 2000).
Because the loss of ush function resulted in a supernumerary
cardial cell phenotype, the effect of expressing the gene throughout the mesoderm was monitored using the Gal4/UAS binary system. Mef2 was used to assess the status of cardial cells, with two contiguous rows of 52 cells present in the forming or mature dorsal vessel of wild-type embryos. In comparably staged embryos expressing
ush throughout the mesoderm, a significant reduction in
cardial cells is observed. The D-mef2 heart enhancer-lacZ fusion gene
was used as a second marker for the cardial cells and also to assay the
effect of ush expression on enhancer activity. In wild-type
embryos at stage 16, the enhancer is active in eight cardial cells in
most segments of the dorsal vessel. In contrast,
beta-galactosidase activity is greatly diminished in the hearts of
ush-expressing embryos, most likely a combination of the
decrease in cardial cell number and the reduced activity of the
D-mef2 cardiac enhancer. Thus, forced
expression of ush has a potent negative effect on cardial
cell formation and enhancer function (Fossett, 2000).
It has been shown that Pnr can function combinatorially with
Tin in the regulation of the D-mef2 heart enhancer in Drosophila embryos. This
synergistic activation was examined using a transient transfection assay in cultured
Drosophila cells. The activation of a CAT reporter gene
linked to the D-mef2 enhancer was monitored in cells
transfected independently with Pnr, Tin, and Ush or with various
combinations of the factors. The expression of Tin alone activated the
enhancer about 2-fold above the basal level, whereas neither Pnr nor
Ush affected enhancer activity. Coexpression of Pnr and Tin resulted in a synergistic
activation of the enhancer to a level 5-6 times that of the basal
activity, and this strong induction requires the
binding of Tin to the Mef2 enhancer; a Tin DNA binding
mutant, Tin (N-Q), failed to synergize with Pnr in the assay. In contrast, adding Ush as a third transfected factor
significantly inhibits the synergistic activation of the enhancer by
Pnr and Tin. This result demonstrates that Ush can
antagonize the positive functional interaction of Pnr and Tin in the
regulation of the cardial cell enhancer, which is consistent with the
in vivo data (Fossett, 2000).
ush mutants contain an increased number
of cardial cells. However, in about half of the embryos
a disparity was noticed in the cardial cell populations, ranging from a high of
8-12 per hemisegment down to regions completely devoid of cells. This
complex phenotype is observed with both ush hypomorphic and null alleles. This sporadic loss of cells from the
dorsal-most part of the mesoderm is reminiscent of a htl
mutant phenotype, where the absence of the encoded fibroblast growth
factor receptor homolog results in an incomplete dorsal migration of
mesodermal cells. In this event, cells fail to receive the
ectodermal signal needed for their further commitment, resulting in a
loss of dorsal mesodermal derivatives, including cardioblasts (Fossett, 2000).
To determine whether the variable absence of cardial cells in
ush embryos is because of a cell migration defect, wild-type and mutant embryos were stained for Mef2 protein present in the invaginated population of mesodermal cells. In cross sections of normal
embryos at stage 10, a uniform layer is observed where the dorsal-most
mesodermal cells have migrated to a position adjacent to the
dorsal-most ectodermal cells. In contrast, both htl and ush homozygous embryos display an irregular layer where mesodermal cells remain clustered and fail to undergo their complete
dorsal migration. This result suggests ush function is
required for the directional migration of the mesoderm. To investigate
a potential genetic interaction of htl and ush in
this process, embryos were examined that were heterozygous for
mutations in each of the genes. These embryos also present a strong
mesodermal migration phenotype, suggesting the two
genes function in a common genetic pathway that controls this aspect of
mesoderm differentiation. As was observed with homozygous
ush embryos, slightly less than half of the transheterozygous embryos show a loss of cells from the dorsal mesoderm (Fossett, 2000).
It is thus thought that pnr has a dual requirement in the cardiogenic mesoderm because it is needed for the formation of cardial cells although
simultaneously limiting the production of Eve-expressing pericardial
cells. Based on these dissimilar phenotypes, it is postulated that Pnr
might work with different combinations of factors to promote or repress the formation of cells within the distinct lineages. Recent studies have shown that Pnr and Tin act synergistically to induce cardial cells
and activate gene expression, and the loss of function of either of
these genes results in an absence of cardial cells. Therefore, the two
work together in cardial cell specification (Fossett, 2000).
In contrast, Ush is a factor that normally suppresses cardial cell
production. ush homozygous and hemizygous embryos show an
increase in cardial cell number: the latter finding suggests Ush
control of this cell population is dose-dependent, as is the case for
Ush regulation of Pnr during sensory bristle development.
Furthermore, forced expression of Ush decreases cardial cell production
and D-mef2 heart enhancer activity, whereas ectopic expression of Pnr produces extra cardial cells and expands the domain
of enhancer activity. Thus, Ush displays phenotypes that are in direct
opposition to those of Pnr, suggesting that it can function as an
antagonist of Pnr's cardiogenic activity. This conclusion is
supported by the ability of Ush to inhibit the synergy of Pnr and Tin
in the activation of the D-mef2 heart enhancer in cell
transfection studies. As for the second cardiac phenotype, both
pnr and ush are required to limit the number of Eve-expressing pericardial cells, consistent with a model in which Ush
and Pnr function as corepressors in the control of these cells. To summarize, these genetic studies predict that, in the wild-type
embryo, pnr is expressed and functions independent of
ush in precursors of the cardioblast lineage. However, in
neighboring cells that include the Eve lineage precursors, the
expression and function of the two most likely overlaps. Future
expression analyses of the two regulatory proteins at the resolution of
single mesodermal cells will be required to elaborate on this genetic model in molecular terms (Fossett, 2000).
The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When U-shaped (Ush), FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. Ush appears to negatively regulate the cardiogenic function of the GATA-4 homolog Pnr, converting Pnr from a transcriptional activator to a repressor as observed during sensory bristle development. As with Ush, forced mesodermal expression of FOG-1, FOG-2, and DeltaFOG-2 (lacking a conserved motif that binds the corepressor C-terminal binding protein) also produces a diminution of cardial cells. These results demonstrate a functional conservation of the FOG proteins during Drosophila cardiogenesis, which most likely involves negative regulation of the cardiogenic activity of Pnr. In addition, forced expression of FOG proteins disrupts eye development-producing phenotypes that mimic pnr loss of function mutants, presumably by repressing Pnr activation of its downstream effector genes (Fossett, 2001).
Inductive signaling is of pivotal importance in order for developmental patterns to form. In Drosophila, the transfer of TGFß (Dpp) and Wnt (Wg) signaling information from the ectoderm to the underlying mesoderm induces cardiac-specific differentiation in the presence of Tinman, a mesoderm-specific homeobox transcription factor. Evidence that the Gata transcription factor, Pannier, and its binding partner U-shaped, also a zinc-finger protein, cooperate in the process of heart development. Loss-of-function and germ layer-specific rescue experiments suggest that pannier provides an essential function in the mesoderm for initiation of cardiac-specific expression of tinman and for specification of the heart primordium. u-shaped also promotes heart development, but unlike pannier, does so only by maintaining tinman expression in the cardiogenic region. By contrast, pan-mesodermal overexpression of pannier ectopically expands tinman expression, whereas overexpression of u-shaped inhibits cardiogenesis. Both factors are also required for maintaining dpp expression after germ band retraction in the dorsal ectoderm. Thus, it is proposed that Pannier mediates as well as maintains the cardiogenic Dpp signal. In support, it is found that manipulation of pannier activity in either germ layer affects cardiac specification, suggesting that its function is required in both the mesoderm and the ectoderm (Klinedinst, 2003).
pnr and ush are both expressed in the mesoderm at the
time of cardiac mesoderm formation, in addition to their expression in the dorsal ectoderm.
Mesodermal expression of pnr is restricted to the dorsal cardiogenic
margin, whereas ush extends more laterally. In
order to assess the requirement for pnr and ush in
initiating cardiac mesoderm and cardiac cell type-specific differentiation, tin expression was examined at progressively later developmental
stages in null mutants for both pnr and ush. During
mid-stage 11, tin is expressed segmentally in two regions of the
mesoderm. The dorsal
clusters of cells correspond to the cardiac precursor cells, whereas the lateral clusters will become part of the visceral mesoderm. In same stage pnr mutant embryos, tin expression is dramatically reduced in the clusters that correspond to the cardiac precursors, indicating that
cardiogenesis is not being initiated. tin expression in the visceral mesodermal
clusters, as well as tin expression earlier in development, is
unaffected, suggesting the heart is a focal point for pnr function, which is consistent with its cardiac-restricted expression in the mesoderm. By
contrast, ush mutant embryos initially seem to exhibit normal
tin expression. At later stages, when tin expression is solely restricted to the heart cells, ush mutants display a progressively more severe reduction in tin expression, approaching the phenotype of
pnr mutants. Thus, both pnr and ush are required for heart-specific tin expression, although ush seems to be initially dispensable (Klinedinst, 2003).
Even though tin is initially expressed in all heart progenitors, its expression is later turned off in some specific lineages, but continues to
be expressed in many myocardial and pericardial cells. To
determine which heart cells are affected in pnr and ush
mutants, mutant embryos were examined with various markers. eve, for example, is co-expressed with tin in 11 clusters of heart progenitors, and these lineages give rise to a subset of pericardial cells. eve expression is only moderately reduced in pnr and hardly at all in ush mutants at early as well as later stages; this is accompanied by patterning defects at progressively later stages. By contrast, the lbe-expressing heart progenitors, which produce both myocardial and pericardial cells, are dramatically reduced in pnr but less so in ush mutants. Moreover, the svp-expressing cells, which also give rise to a mixed lineage, but cease to co-express tin at later
stages, are dramatically reduced in both mutants. Thus, all lineage
markers assayed are reduced in both mutants, but each is affected with
disproportional severity, which is consistent with the idea that the formation of each cell type has a direct requirement for pnr and
ush (Klinedinst, 2003).
Both tin and pnr have been shown to be targets
of Dpp signaling at stage 9/10. It is proposed that dpp is necessary again at stage 11 to activate and maintain pnr and tin expression in the cardiogenic region of the mesoderm. First, pnr is activated with the help of early stage
11 tin, which is expressed broadly throughout the dorsal mesoderm, and dpp, which is expressed in a narrow dorsal ectodermal stripe. Then, at mid-stage 11, tin is restricted to the cardiogenic region with the help of mesodermal pnr as well as continuous ectodermal Dpp signaling. Once both are activated in the cardiogenic mesoderm, they are likely to contribute to the maintenance of each other's expression, probably aided again, but only moderately, by ectodermal Dpp signaling. This interpretation is consistent with mesodermal versus ectodermal expression of dominant-negative pnrEnR and the dpp target repressor encoded by brk. They are both equally effective in reducing cardiac-specific tin when expressed in the mesoderm, but ectodermal repression is more effective when dorsal-stripe dpp at stage 11 is also affected (as in the case of ZKr-Gal4>UAS-brk, but not with ZKr-Gal4>UAS-pnrEnR) (Klinedinst, 2003).
Mesodermal overexpression of ush and co-overexpression with
pnr results in a decrease in the amount of cardiac-specific
tin expression, suggesting that ush may not only be required along with pnr for heart development, but also play an inhibitory role. To test this hypothesis further, pnrD4, an
allele that abolishes Ush binding to Pnr was overexpressed; not only ectopic tin expression was found at early stages of cardiogenesis, but also
undiminished and even increased levels of expression at later stages. A
similar phenotype was observed when both pnrD4 and ush were expressed throughout the mesoderm, suggesting that ush plays an anti-cardiogenic role by antagonizing the activity of wild-type Pnr, but not that of PnrD4. It would be interesting to see if pan-mesodermal overexpression of wild-type pnr in a ush mutant background results in
ectopic tin expression similar to pnrD4, or if a minimal
amount of ush activity is required to maintain normal and ectopic tin expression even with forced pnr expression.
Interestingly, overexpression of both pnr and tin together in the mesoderm also causes a pnrD4-like phenotype, as assayed with Hand expression, suggesting that pnr and tin collaborate during initiation and subsequent differentiation of the heart progenitors (Klinedinst, 2003).
Although in vitro the Ush-related FOG factors are primarily known for their role as transcriptional repressors, they
apparently can also function as co-activators: Fog2 can synergistically
activate or repress the transcriptional activity of Gata4, depending on the (cardiac) promoter and cell line used, and FOG-1 can cooperate with Gata1 to transactivate
NF-E2, an erythroid cell-specific promoter.
Moreover, the ventricular hypoplasia and other heart defects observed in Fog2-deficient mice suggest a deficit rather than an excess in heart
development. In addition, mice with an equivalent mutation to PnrD4
knocked into the Gata4 locus, thus eliminating binding to Fog2, exhibit in many ways a similar phenotype to Fog2-deficient mice. These
data are consistent with the idea that Fog2 is normally involved in promoting rather than antagonizing cardiogenesis, similar to what was found with genetic studies during Drosophila heart development (Klinedinst, 2003).
The dual role of Ush suggests that the amount of Ush may be crucial for whether it exerts its function as a an activator or repressor, perhaps by binding to different sets of co-factors in a concentration-dependent manner. Alternatively, the mode of transcriptional regulation by Ush could be stage-dependent: at stage 11, Pnr and Ush cooperate as transcriptional activators in initiating cardiac-specific tin expression and heart development, but later Ush becomes a repressor to limit the transcriptional
activation of tin by Pnr (Klinedinst, 2003).
pnr and ush are initially broadly expressed in the dorsal ectoderm of the early embryo, but by germband retraction the ectodermal expression of pnr is confined to a narrow stripe of cells along the border of the amnioserosa, which overlaps with the thin dorsal dpp stripe. The early
ectodermal expression of ush is restricted to the presumptive
amnioserosa, and by germband extension, ush also overlaps with the dorsalmost region of the ectoderm. These patterns of expression suggest that pnr and ush may be acting in both germ layers. The genetic data, including germ layer-specific expression of wild-type and dominant-negative pnr constructs, as well as germ layer-specific rescue experiments suggest strongly that pnr and ush function is not only needed in the mesoderm, but also in the ectoderm for heart formation. The ectodermal requirement for pnr and ush in heart development is probably achieved via the maintenance of dpp
expression, since dorsal stripe dpp expression diminishes in
pnr and ush mutants and ectodermal interference with
pnr, ush and/or dpp-signaling function compromises the
normal progression of heart development (Klinedinst, 2003).
The Drosophila heart consists of two major cell types: cardioblasts, which form the contractile tube of the heart; and pericardial cells, which flank the cardioblasts and are thought to filter and detoxify the blood or hemolymph of the fly. This study presents the completion of the entire cell lineage of all heart cells. Notably, a previously unappreciated distinction has been detected between the lineages of heart cells located in the posterior seven segments relative to those located more anteriorly. Using a genetic screen, the ETS-transcription factor pointed has been detected as a key regulator of cardioblast and pericardial cell fates in the posterior seven segments of the heart. In this domain, pointed promotes pericardial cell development and opposes cardioblast development. This function of pointed is carried out primarily if not exclusively by the pointedP2 isoform -- in this context, pointedP2 may act independently of Ras/MAPK pathway activity. The GATA transcription factor pannier acts early in dorsal mesoderm development to promote the development of the cardiac mesoderm and thus all heart cells. Finally, it is demonstrated that pannier acts upstream of pointed in a developmental pathway in which pannier promotes cardiac mesoderm formation, and pointed acts subsequently in this domain to distinguish between cardioblast and pericardial cell fates (Alvarez, 2003).
Pioneering work in C. elegans established the importance of elucidating cell lineages to obtain a thorough understanding of animal development. Prior work established the cell lineage of 10 out of the 16 heart cells that arise in each hemisegment of the posterior seven heart segments. To define the lineage of the remaining heart cells, the FLP/FRT lineage tracing system was used to determine the lineal relationship of the two Eve-positive pericardial cells and four Tin-positive pericardial cells that arise in each hemisegment. This system creates random clones marked by tau-lacZ reporter gene activity. Briefly, clones were induced during stage 8 just as the pan-mesodermal divisions are being completed. This allowed clones to be induced in mesodermal cells prior to the emergence of heart precursors. To identify the lineage of Eve-positive pericardial cells, embryos were double labeled for ß-galactosidase to mark clones, and with Eve to identify Eve-positive pericardial cells. To identify the lineage of Tin-positive pericardial cells, embryos were double labeled for ß-galactosidase to mark clones, and Tin to identify Tin-positive pericardial cells. In addition to the Tin-positive pericardial cells, Tin labels cardioblasts and Eve-positive pericardial cells. However, based on position and morphology one can unambiguously distinguish Tin-positive pericardial cells from Tin-positive cardioblasts and Eve-positive pericardial cells (Alvarez, 2003).
Eighteen clones were identified that contained at least one Eve-positive pericardial cell. Eleven of these clones (61.1%) consisted solely of two Eve-positive pericardial cells, six clones consisted of two Eve-positive pericardial cells and one or two nearby heart or other mesodermal cells, and one clone consisted of a single Eve-positive pericardial cell. Thus, when one Eve-positive pericardial cell is observed within a clone of two or more cells a second Eve-positive pericardial cell always exists within this clone. These data demonstrate that the two Eve-positive pericardial cells within a hemisegment are siblings and arise from an Eve-positive pericardial cell precursor (Alvarez, 2003).
Tin-positive pericardial cell clones fall into two classes: those that contained two Tin-positive pericardial cells, and those that contained one Tin-positive pericardial cell and one cardioblast. These two classes of clones arise in mutually exclusive regions of the heart. Clones that contain two Tin-positive pericardial cells arise in the posterior seven segments of the heart (this region is referred to as the posterior heart domain), whereas clones that contain one Tin-positive pericardial cell and one cardioblast arise anterior to this domain (this region is referred to as the anterior heart domain). The point of demarcation between these clonal types coincides precisely with the location of the first pair of Svp-positive cardioblasts. These data demonstrate that heart cells exhibit distinct cell lineages as a function of position along the anteroposterior axis (Alvarez, 2003).
A total of 24 Tin-positive pericardial cell clones were identified in the posterior heart domain. Fifteen of these clones consisted solely of two Tin-positive pericardial cells, eight clones consisted of two Tin-positive pericardial cells and two nearby mesodermal cells, and one clone consisted of a single Tin-positive pericardial cell. Thus, when one Tin-positive pericardial cell is observed within a clone of two or more cells, a second Tin-positive pericardial cell always exists within this clone. These data indicate that the four Tin-positive pericardial cells found in each hemisegment of the posterior domain arise from two Tin-positive pericardial cell precursors. The inability to identify any clones that contain four Tin-positive pericardial cells indicates that adjacent Tin-positive pericardial cell precursors are unlikely to share a common lineage (Alvarez, 2003).
Eighteen Tin-positive pericardial cell clones were identified in the anterior heart domain. All 18 clones consisted of one Tin-positive pericardial cell and one cardioblast. These data indicate that within this region Tin-positive pericardial cells arise from bi-potent heart precursors, each of which produces one Tin-positive pericardial cell and one cardioblast. These data also demonstrate that cardioblasts and Tin-positive pericardial cells in the anterior heart domain develop via a different cell lineage than cardioblasts and Tin-positive pericardial cells that develop in the posterior domain (Alvarez, 2003).
The analysis of ten additional cardioblast clones in the anterior heart domain support a distinct cell lineage for anterior versus posterior cardioblasts. Nine clones consisted of one cardioblast and one non-Tin-expressing pericardial cell, whereas a single clone consisted of two cardioblasts. Thus, most, if not all, anterior domain cardioblasts share a sibling relationship with a pericardial cell. In addition, all anterior domain cardioblasts exhibit cell lineages distinct from posterior domain cardioblasts. Together with the lineage data on Tin-positive pericardial cells, these results support the idea that cardioblasts and Tin-positive pericardial cells in the anterior heart domain carry out distinct functions from those found in the posterior heart domain (Alvarez, 2003).
Interestingly, the lineage of the twelve cardioblasts in the anterior heart domain appears fixed with respect to whether they share a sibling relationship with a Tin-positive or Tin-negative pericardial cell. These cardioblasts were numbered 1-12 from anterior to posterior with cardioblast 12 being immediately anterior to the first Svp-positive cardioblast. Four clones were identified that contained cardioblast 12 and in each clone this cardioblast shared a sibling relationship with a Tin-negative pericardial cell. By contrast, cardioblasts 10 and 11 each share a sibling relationship with a Tin-positive pericardial cell. No multiple clones were obtained for all twelve cardioblasts; nonetheless, these data suggest a fixed relationship between the position of a cardioblast and whether its sibling pericardial cell expresses Tin. It is speculated that the differences in gene expression between different pairs of sibling cardioblasts and pericardial cells in the anterior domain may reflect functional differences between such pairs of heart cells (Alvarez, 2003).
pnt was identified as an inhibitor of cardioblast development in a
screen for mutations that affect cardioblast and/or pericardial cell
development. To identify genes that regulate heart development
~2000 third chromosomal lethal P element lines were screened for defects in the expression of Mef2 a protein
expressed in all cardioblasts and Eve. Two P element mutations were uncovered that cause an approximate twofold increase in cardioblasts. One of these P elements [l(3)S012309] maps to
cytological position 94F1-3 and was known to be an allele of pnt. To verify that
lesions in pnt result in the formation of ectopic cardioblasts, the phenotype of five additional pnt alleles was assayed. Although the severity of the phenotype varies for each pnt allele, all alleles display a significant increase in cardioblast number relative to wild-type embryos. With respect
to the excess cardioblast phenotype, these alleles can be grouped into an allelic series. The presence of excess cardioblasts in embryos homozygous mutant for each pnt allele indicates that pnt normally functions in heart development to repress cardioblast development (Alvarez, 2003).
The published heart phenotype of the GATA transcription factor pnr is opposite that of pnt. In pnr mutant embryos, too many pericardial cells and too few cardioblasts are thought to develop. As a first step towards examining the potential regulatory interactions between pnr and pnt, a detailed analysis was carried out of heart development in pnr mutant embryos. pnrVX6, a null allele that contains a small deletion that removes all but the N-terminal nine amino acids of pnr, was used as well as pnr1, a molecularly uncharacterized allele. In contrast to a prior study, a loss of both cardioblasts and pericardial cells was found in pnr embryos.
The dorsal mesodermal phenotypes were quantified for Eve-positive pericardial cells as well as for all pericardial cells using the pan-pericardial marker Zfh1. In wild-type embryos an average of 22.7 Eve-positive pericardial cells and 61.1 Zfh1-positive pericardial cells were observed per embryo side. pnrVX6 embryos exhibit
the most severe effect with an average of 9.4 and 16.9
Eve- and Zfh1-positive pericardial cells, respectively.
pnrVX6/pnr1 embryos exhibit an intermediate
phenotype with an average of 16.4 Eve-positive pericardial cells
and 27.4 Zfh1-positive pericardial cells, while
pnr1 embryos exhibit the mildest phenotype with an average of 21.2 and 37.4 Eve- and Zfh1- positive
pericardial cells, respectively. A severe loss of
cardioblasts and Odd-positive pericardial cells were observed in these backgrounds although these phenotypes were not quantified. The loss of cardioblasts and Odd-positive pericardial cells is most severe in pnrVX6 embryos and least severe in pnr1 embryos where short
stretches of cardioblasts are still visible. These results
indicate that pnr normally functions to promote the development of all heart cells. The results demonstrate that the earliest manifestation of cardiac mesoderm development is defective in pnr embryos, suggesting that the general lack of heart cells in pnr embryos arises indirectly via a defect in the specification of the cardiac mesoderm. Double mutant studies support the model that pnr acts upstream of pnt in a developmental pathway (Alvarez, 2003).
This paper indicates that pnr and pnt act
sequentially to regulate heart development.
pnr acts early in mesoderm development to enable the cardiac mesoderm to form. Subsequent to this event, pnt acts within the cardiac mesoderm to regulate the ability of cells to choose between the pericardial or cardioblast fate. In this context, pnt inhibits the development of the Svp-class of cardioblasts and appears to function independently of Ras/MAP kinase pathway activity (Alvarez, 2003).
The effect of pnt on heart development is restricted to the
posterior seven heart segments where Svp cardioblasts normally develop.
Interestingly, the lineage studies identify a clear difference in the cell lineage of cardioblasts that develop in the posterior seven heart segments versus those that develop more anteriorly. These
results identify a genetic and developmental distinction between these two regions of the heart. In addition, they suggest that cells in different regions of the heart carry out different functions and that these functions are probably under homeotic gene control. Future work that addresses the physiological role of these cells in heart function and the control of their development by homeotic genes should provide a more comprehensive understanding of heart development (Alvarez, 2003).
The data suggest that PntP2 may regulate cardioblast and pericardial cell development independently of Ras/MAP kinase activity. Given that every other developmental function of pnt has been traced back to receptor tyrosine kinase/Ras signaling activity, the apparent Ras independent activity of PntP2 is puzzling. Since PntP2 is expressed broadly throughout the mesoderm, a number of models can explain the apparent Ras-independent activity of PntP2 in the heart. For example, PntP2 may not require MAP-kinase-mediated phosphorylation to carry out a subset of its function. Consistent with this, phosphorylation of PntP2 does not appear to affect its DNA-binding ability. Thus, in the absence of MAP-kinase stimulation, PntP2 is still probably able to bind target promoters alone or in complexes with other proteins. Such an activity of PntP2 could on its own regulate target gene expression by blocking the ability of other transcriptional effectors to bind to and activate target gene transcription, or through an obligate association with other proteins required to activate (or to repress) target genes (Alvarez, 2003).
A second model is that PntP2 requires MAP kinase activation but that this
activity is carried out by one of the other MAP kinase pathways in
Drosophila: the JNK pathway or the p38 pathway. Preliminary
phenotypic analyses indicate that heart development is normal in embryos
mutant for basket, the Drosophila JNK-kinase. Analysis of p38 kinase activity is presently limited because of
the absence of suitable genetic backgrounds. A third possibility is that a novel Ras-dependent pathway does in fact activate PntP2 during heart
development. This model is consistent with the recent identification of a novel receptor tyrosine kinase expressed in the developing visceral mesoderm. Experiments that failed to identify a pnt-like excess cardioblast phenotype upon mesodermal overexpression of a dominant-negative form of Ras argue against this model. However, Ras is maternally loaded and it is extremely difficult to eliminate all Ras activity in this manner. Thus, even though Ras-like mesodermal phenotypes were observed in these experiments, a role for Ras in regulating cardioblast number may have been missed because
of differential sensitivity of different developmental pathways to partial Ras inactivation. Future work that (1) addresses the ability of MAP-kinase insensitive forms of PntP2 to regulate heart development, and (2) identifies PntP2 target genes in the heart and elucidates how PntP2 regulates such genes should help clarify the molecular basis through which PntP2 governs heart development (Alvarez, 2003).
Phenotypic analysis of pnr conflicts with a prior study that
showed an increase in pericardial cells in pnr mutants. This
study used Eve to identify a subset of pericardial cells in
pnr1 embryos. The difference in results are attributed
to use of the pnrVX6 null allele, the ability to
distinguish unambiguously Eve-positive pericardial cells from Eve-positive
somatic muscle progenitors, and to specific defects in dorsal closure
exhibited by pnr embryos that result in the local aggregation of
cells in the dorsal region of the embryo. Genetic results identify
pnr1 as a hypomorphic allele and Eve-positive
pericardial cell formation is found to be almost wild type in this background. In these
experiments, Eve-positive pericardial cells were unambiguously identified via
their co-expression of Zfh1 and it was possible to quantify precisely
Eve-positive pericardial cell number in pnr1 embryos. This
is important as one can observe local increases in Eve-positive mesodermal cells in pnr embryos. However, such apparent increases arise from the local aggregation of dorsal mesodermal cells in pnr1 embryos caused by defects in dorsal closure and not by an overall increase in
Eve-positive mesodermal cells (Alvarez, 2003).
The genetic identification of pnr1 as a hypomorphic
allele is intriguing given that molecular and expression analyses indicate the pnr1 lesion results from a premature stop codon in the middle of the first zinc finger and that the Pnr1 protein localizes
predominantly to the cytoplasm. This lesion is expected to abrogate the
DNA-binding ability of the Pnr protein. However, genetic experiments
indicate that the Pnr1 protein retains residual activity at least with respect to heart development. These results raise the possibility that Pnr may be able to carry out some of its functions independently of DNA binding. Future
work that focuses on a detailed structure function analysis of the Pnr protein should clarify whether Pnr can act independently of its DNA-binding ability in some developmental contexts (Alvarez, 2003).
The pnt allelic series indicates that
pntDelta88 exhibits a milder
excess cardioblast phenotype than pntS012309,
pnt2, and pntRR112. This result is
surprising as pntDelta88
deletes the exons pntP2 shares with pntP1 and as a result
pntDelta88 is assumed to be an amorphic allele of the pnt locus. Using
antisense RNA probes specific for the unique exons of pntP2, an essentially wild-type pattern of pntP2 transcription is observed in
pntDelta88 mutant embryos. These data raise the possibility that the N-terminal regions
of pntP2 may also retain partial activity. Studies along the lines of those suggested for Pnr should also help elucidate whether truncated forms of PntP2 retain residual activity (Alvarez, 2003).
Significant similarity exists between the embryology and
molecular regulation of early heart development in Drosophila and
vertebrates. In this context, the identification of a role for pnt (a member of the evolutionarily conserved ETS transcription factor family) in Drosophila heart development raises the possibility that ETS family proteins regulate vertebrate heart development. Consistent with this, ETS1 and ETS2, the two most closely related vertebrate ETS proteins to pnt, are expressed in the developing vertebrate heart -- functional studies indicate these genes regulate the expression of specific genes in the heart. However, knockout studies have not yet revealed a clear role for ETS1 or ETS2 in the morphological development or differentiation of the vertebrate heart. The existence of multiple vertebrate ETS-family members highly homologous to pnt, as well as a total of 25 ETS family members in humans suggests the possibility of functional redundancy in ETS protein function during vertebrate and mammalian heart development. Thus, a full understanding of ETS protein function during heart development awaits construction and analysis of animals multiply mutant for different ETS family members (Alvarez, 2003).
Dorsoventral (DV) patterning is essential for growth of the Drosophila eye. Recent studies suggest that ventral is the default state of the early eye, which depends on Lobe (L) function, and that the dorsal fate is established later by the expression of the dorsal selector gene pannier (pnr). However, the mechanisms of regulatory interactions between L and dorsal genes are not well understood. For studying the mechanisms of DV patterning in the early eye disc, a dominant modifier screen was performed to identify additional genes that interact with L. The criterion of the dominant interaction was either enhancement or suppression of the L ventral eye loss phenotype. Forty-eight modifiers were identified that corresponded to 16 genes, which included fringe (fng), a gene involved in ventral eye patterning, and members of both Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways, which promote L function in the ventral eye. Interestingly, 29% of the modifiers (6 enhancers and 9 suppressors) identified either were known to interact genetically with pnr or were members of the Wingless (Wg) pathway, which acts downstream from pnr. The detailed analysis of genetic interactions revealed that pnr and L mutually antagonize each other during second instar of larval development to restrict their functional domains in the eye. This time window coincides with the emergence of pnr expression in the eye. These results suggest that L function is regulated by multiple signaling pathways and that the mutual antagonism between L and dorsal genes is crucial for balanced eye growth (Singh, 2005).
Axial patterning plays a crucial role in organizing growth and in differentiating developing fields. To understand how the DV pattern is established in the Drosophila eye, the genetic relationships between dorsal and ventral eye genes were analyzed. A group of new genes was identified that modifies the L mutant eye phenotype not only by misexpression but also by reduced gene function (Singh, 2005).
In the early eye disc, fng is preferentially expressed in the ventral eye. The DV domain specification by Fng is also important for growth of the eye disc as its ubiquitous overexpression in the eye disc blocks eye development. Even though L and fng play important roles during ventral eye growth and patterning, the developmental interaction between the two has been unknown. This study showed that overexpression of fng can partially compensate for the loss of L gene function in the eye. This suggests that fng works either downstream or parallel to L in the growth of the ventral eye. It is possible that L and fng interact through the induction of a common target, Ser, in the eye (Singh, 2005).
Like several other pathways, Wg signaling has multiple functions during eye development. This study identified Sgg, a serine/threonine kinase, as a modifier that suppresses the L mutant phenotype upon overexpression. Sgg is known to inhibit the Wg signaling pathway by downregulating Armadillo (Arm) via ubiquitin-mediated proteosomal degradation. Other components of the Wg signaling pathway such as pygo and dally were also identified as modifiers, which, upon overexpression in the eye, enhanced the L mutant phenotype. These results suggest that Wg signaling acts antagonistically to L function in the ventral eye. The genetic interaction of these EP lines with the L mutations represents specific enhancement rather than additive effects, since antagonists of Wg signaling were identified as suppressors, whereas members required for Wg signaling were identified as enhancers of the L mutant phenotype in the EP screen (Singh, 2005).
In this screen, it was found that the overexpression of Daughters against Dpp (Dad), an antagonist of Dpp signaling, enhances the L mutant phenotype, whereas EP insertions at hh and its receptor gene smo were identified as suppressors of the L mutant phenotype. The members of these two signaling pathways are known to be involved in eye growth and differentiation. These results raise another interesting possibility of the possible role of Hh and Dpp signaling pathways in early eye growth and patterning (Singh, 2005).
During Drosophila eye development, Hh controls progression of the furrow by inducing the expression of dpp and atonal (ato), a proneural gene responsible for R8 photoreceptor formation. In the eye, hh and dpp are involved in a positive feedback loop for the initiation and movement of the MF whereas Wg signaling acts antagonistically to Dpp signaling to block MF movement and progression. This antagonistic relation may be present even during early eye development since wg and dpp are localized to opposing regions of the undifferentiated younger eye primordia: dpp along the posterior margin and wg across the dorsal anterior region. These results suggest that the early function of Hh and Dpp signaling is to promote L-mediated ventral eye growth whereas Wg signaling acts as an antagonist (Singh, 2005).
BarH1 and BarH2 were identified as enhancers of the L mutant phenotype. BarH1 and BarH2 are a pair of homeobox proteins that express in a subset of photoreceptors and in the basal undifferentiated cells of the eye disc. B is required for the negative regulation of eye development by repressing the expression of the proneural gene ato. However, it is not known whether B plays a role in early eye growth, prior to retinal differentiation. Clonal analysis has not yet revealed evidence for B function in DV asymmetric eye patterning. However, the current data showed genetic interactions of L mutants with GOF and LOF mutants of B. The suppression of the L2/+ eye phenotype by a LOF mutation of B, which by itself has no defects in the eye, raises the possibility that B itself may not have DV asymmetric function but needs to be downregulated by L for normal growth of the early eye disc. This is also consistent with the dramatic eye reduction observed when ey-GAL4 drive B is overexpressed during early eye development. It has been shown that Wg and B have both positive and negative regulatory relationships in prepatterning of the notum. B expression is activated by Wg in the scutum whereas B represses Wg expression in the most anterior part of the notum. L mutants respond to GOF and LOF of both Wg signaling and B in a similar fashion, suggesting that Wg and B may be regulating each other positively during early eye development (Singh, 2005).
L is known to act downstream of N. In the eye, emc and h, the repressors of ato, are downregulated by N. During eye development, emc acts in collaboration with hairy (h) as the negative regulator of the morphogenetic furrow by repressing ato. Therefore, identification of emc as an antagonist of L-mediated early ventral eye growth seems possible. Interestingly, both emc and B have also been identified as modifiers of pnr (Singh, 2005).
Some of the genes that were identified as L modifiers, such as B, emc, and smo, have been well characterized, but their roles in early eye disc growth and/or DV asymmetric function have not been studied. Genes were identified involved in cell survival and growth such as disc over grown (dco), a member of the serine/threonine protein kinases family, and genes involved in vesicular trafficking, including RhoGAP68F, an ion transport such as nrv 1, and the acetyl transferase nej. It is possible that potential DV asymmetric function of these genes might have been missed by LOF analysis because of functional redundancy or these genes may be modifying the early growth function of L in the eye. More in-depth studies will be necessary to explore these possibilities. However, it is important to note that both GOF and LOF of these genes exhibit specific genetic interactions with L mutant backgrounds. In addition to the well-characterized genes, a few novel genes like EP1229 and EP1595 were identified whose functions are not known. These genes were not listed in this study as the specificity of their genetic interaction with L was not tested by using LOF mutations (Singh, 2005).
The results demonstrate that the level of pnr gene function is a crucial factor for DV patterning of the eye as increased levels of pnr gene function enhance the L mutant phenotype of ventral eye loss to no eye, whereas reduction of pnr gene function rescued the loss of the ventral eye phenotype of the L mutant. Further, the phenotypes of LOF clones of L where only the ventral cells are lost can be rescued by reducing the levels of pnr gene function. These results suggest that pnr acts antagonistically to the ventral eye growth function of L. However, it was also found that the antagonism of pnr and L is mutual. This conclusion is based on the fact that the gain-of-function phenotype of pnr in the eye is significantly enhanced when L function is reduced. These conclusions were also validated by showing that the dorsal eye enlargements associated with LOF clones of pnr can be prevented by reducing the levels of L gene function. These results suggest that optimal levels of pnr and L are necessary for DV patterning and growth of the eye. It was also found that the downstream dorsal eye selectors, Iro-C members (ara, caup, mirr) are involved in a mutually antagonistic relationship with L. These studies demonstrate that the antagonism of L holds true for key components involved in dorsal fate selection during early eye development (Singh, 2005).
The time window of the second instar of larval development was identified as the periord during which mutual antagonistic interaction of L and pnr is required for DV patterning and growth in the eye. Previously, it was shown that the pnr function in eye development is critically required during the second instar larval stage. This time window is coincident with the one that is required for the antagonism of pnr and L as shown in this study, suggesting that a major function of L in early eye development is to establish the DV domains by negatively regulating the dorsal selectors. These studies also support the physiological relevance of this mutually antagonistic interaction in DV patterning (Singh, 2005).
It is not known how L antagonizes Pnr function. One possibility is that L may be required for restricting the pnr expression domain to the dorsal margin of the eye disc. It was difficult to check whether L is cell-autonomously required for pnr repression because LOF clones of L result in the elimination of the entire or ventral eye, depending on the time when the clones are generated. Alternatively, the effect of a L mutation on pnr expression was studied. Interestingly, pnr expression, which is restricted to the dorsal eye margin in wild type eye discs, shows a nearly twofold expansion in L2/+ mutant discs. It remains to be studied whether L is required for the repression of pnr expression or for the inhibition of growth of pnr-expressing cells. On the basis of these data it is suggested that during early DV patterning, the onset of pnr expression might restrict the functional domain of L and Ser to the ventral eye. It is possible that pnr may also suppress L gene function via the Wg signaling pathway (Singh, 2005).
The results support the view that various developmental pathways cross-talk with each other to define the final form of a developing eye field. Such genes are likely to interact with both pnr and L. It is interesting to note that several pnr-interacting genes were identied as L modifiers in the screen. This illustrates the importance of the interaction of L and pnr pathways and also the efficacy of the screen. Further study of new modifiers of L may provide important clues to the mechanism of pnr-L interactions in the control of growth and/or DV patterning of the eye. Since the compound eye of Drosophila shares some similarities with the vertebrate eye and genetic machinery is highly conserved, it would be interesting to see if these antagonistic interactions between the dorsal eye selectors and the ventral eye genes play roles in the DV patterning and growth of vertebrate eyes (Singh, 2005).
In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. This study analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate (Mann, 2009).
The specification of a subset of mesodermal cells towards a cardiac fate
requires well-orchestrated interactions of a plethora of factors. Drosophila is the model system of choice to decipher the complex transcriptional network that initiates and sustains a cardiac lineage. The data place tup as an essential component in the early transcriptional network that specifies cardiac mesoderm (Mann, 2009).
After the initially broad expression domain of Tin has become restricted to
the dorsal mesodermal margin, Tup expression is first seen in the cardiac
mesoderm in ~10 small clusters, which co-express Eve. Slightly later, Tup
is present throughout the Tin-positive cardiac mesoderm and gene expression
analyses in tupisl-1, tin346,
pnrVX6 and Df(3L)DocA embryos demonstrate that all
four factors are required to maintain each other's expression. Additionally,
analyses of cardiac gene expression in embryos that are transheterozygotic for
tup and tin, pnr or Doc, showed that these factors
interact genetically to specify heart cells (Mann, 2009).
Although it might be expected that Tup expression is lost in tin
mutants since these embryos are devoid of heart cells, it is interesting that Tup
expression in the early cell clusters is still initiated. This finding is
somewhat reminiscent of the observation that the initiation of Doc expression
is also independent of tin. According to the temporal appearance of Tup in
the cardiac mesoderm with respect to Tin and Doc, tup is required for
their maintenance rather than their initiation. By contrast, the onset of
mesodermal Pnr and Tup expression appears to coincide. It was
not resolved whether Tup is induced by Pnr or directly by Dpp. A direct
regulation by Dpp was implicated by the reduced expression of Tup after
mesodermal overexpression of UAS-brinker, which is
known to bind to dpp-response elements of dpp target genes.
Conversely, it was shown that dpp expression depends on tup and
the present data suggest that this regulation requires pnr (Mann, 2009).
Germ layer-specific inhibition of Tup using a construct that lacks the
homeodomain, but contains the two LIM domains, revealed that Tup can regulate
cardiogenesis in the mesoderm as well as from the ectoderm. Since the
69B-Gal4 driver has been reported not to be strictly ectodermal,
it is possible that mesodermal Tup function was also interfered with. However,
the mesodermal expression of 69B-Gal4 seems to be negligible. The
effect of ectodermal Tup inhibition on cardiogenesis in the mesoderm can only
be explained if the function of a secreted growth factor is impaired. dpp expression was analyzed, and a slight downregulation of its
transcripts was observed in embryos expressing UAS-tupδHD in the
ectoderm. Since this effect might not be sufficient to account for the strong
Tin phenotype, further experiments will be required to determine whether
additional growth factors are affected (Mann, 2009).
To better determine the germ layer-specific contribution of Tup in
cardiogenesis, attempts were made to rescue the Tin phenotype by co-expressing the
full-length tup cDNA. Somewhat unexpectedly, a better
rescue was obtained when both constructs were expressed in the ectoderm rather than in the
mesoderm. Since the LIM domains present in tupδHD can
sequester LIM-domain-binding proteins, a
simple explanation for this finding is that Tup interacts with proteins that
are present in the mesoderm but not in the ectoderm. It is reasonable to hypothesize that in the mesoderm the LIM domains of tupδHD not only act as a
dominant-negative for Tup, but additionally for another, perhaps as yet unidentified, LIM-domain containing protein. Since it has been shown that Pnr can bind Tup through the LIM domains, it is likely that Pnr function was interfered with by overexpressing UAS-tupδHD. The requirement of the LIM domains for proper cardiac specification is shown by the reduction of Tin-expressing cells after mesodermal expression of the UAS-tupδLIM construct. Further experiments are under way to better resolve the molecular function of Tup in the different tissues (Mann, 2009).
Since the mesodermal expression of UAS-tupδHD
resulted in a strong reduction of Tin-expressing cells at early stages of
cardiac mesoderm formation, it was surprising to observe a rather low
reduction of Dmef2-positive myocardial cells at later stages (15/16). To
exclude the possibility that the twi-Gal4 driver does not
sufficiently express UAS-tupδHD throughout
embryogenesis, this experiment was repeated using the combined mesodermal
driver twi-Gal4; 24B-Gal4. However, the phenotypes were not
enhanced. A time course for Tin expression in these crosses
revealed that Tin appears to recover over time. A similar phenomenon can be
seen in tupisl-1 mutants, although it might not be as
obvious because the mutants also lack ectodermal tup expression. In
any case, the data is suggestive of a different temporal requirement for
tup with respect to tin expression. It is known that
tin expression depends on different transcriptional activation events. Consistent
with the onset of Tup expression in the cardiac mesoderm at mid-stage 11, the
earlier phases of Tin expression are unlikely to depend on Tup. Hence, the
initial Tin expression at stages 8-10 is sufficient to generate a considerable
number of Dmef2-positive myocardial cells at later stages (Mann, 2009).
These analyses further implicate that Tup might act as a transcriptional
activator or repressor depending on the cellular context and on the factors
with which it is co-expressed. This is most strikingly observed with respect
to the Odd-expressing pericardial and lymph gland cells. In tup
mutants, Odd-positive cells are missing in both organs. A similar phenotype is seen when Tup is overexpressed in the mesoderm using the twi-Gal4 driver. The loss of Odd-expressing cells in lymph glands is reminiscent of the phenotype observed in tup mutants, although it is less severe. This differential occurrence of the phenotype
indicates that tup can differentially regulate factors involved in
cardiogenesis versus lymph gland development. This is substantiated by the
finding that mesodermal overexpression of tup
results in an increase in Hand expression in the lymph glands, while Hand
expression throughout the dorsal vessel is only mildly affected. Despite the
loss of Odd-positive cells after early mesodermal tup overexpression,
Tup is required in the pericardial and lymph gland cells at later stages to
maintain Odd expression. Moreover, overexpressing tup in the
pericardial cell lineage yields additional Odd-expressing pericardial cells
and rescues Odd expression in the lymph glands (Mann, 2009).
To obtain more insight into possible functional interactions with other
cardiac transcription factors, tup was overexpressed in combination
with pnrD4. The latter is a highly active variant of
wild-type pnr that contains an amino acid substitution in the
N-terminal zinc finger, which abolishes binding of Ush to Pnr.
Mesodermal overexpression of pnrD4 results in robust
ectopic activation of Tin and embryos co-overexpressing tup and
pnrD4 exhibit the same phenotype. Most likely, a possible
influence of Tup on Pnr activity, regardless of whether it is positive or
negative, is concealed by the strong gain-of-function pnr allele.
However, analysis of Eve expression does provide insight into possible
regulatory interactions between Tup and Pnr. Mesodermal overexpression of each
factor alone yields opposing phenotypes, and when both factors are
co-overexpressed PnrD4 can efficiently counteract Tup activity and
prevent the overspecification of Eve cells. Vice versa, Tup can, although only
moderately, counteract the effect of PnrD4. It has been shown that
during patterning of the thorax, Tup can antagonize the proneural activity of
Pnr by forming a heterodimer, and that the physical interaction between Pnr
and Tup is mediated by the two zinc fingers of Pnr. Hence, the somewhat weak, but possibly antagonistic, function of Tup towards PnrD4 in Eve-positive cell specification could be due to the amino acid substitution encoded in the pnrD4 allele, which might weaken the interaction between the two factors, as compared with wild-type
Pnr. Overexpression of a Tup construct that lacks both LIM domains did not
result in expanded Eve-positive clusters, which strongly
suggests that the effect of Pnr on Tup activity, as seen when both factors are
co-expressed, requires the presence of the LIM domains (Mann, 2009).
In summary, these data demonstrate the crucial role of tup in the
proper specification of cardiac mesoderm in an invertebrate organism.
Therefore, tup/Isl1 should be added to the core set of
ancestral cardiac transcription factors. Consequently, this implicates that
the evolution of the vertebrate four-chambered heart does not necessarily
require the acquisition of a novel network of cardiac transcription factors.
At least, it is unlikely that tup/Isl1 is part of a
regulatory network separate from that of tin/Nkx2.5,
pnr/Gata4 and Doc/Tbx5/6 because it is an essential factor for the formation of the simple linear heart tube in the fly (Mann, 2009).
The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, this study demonstrates that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, it was determined that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. It is concluded that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis (Xu, 2012).
Proper dorsal vessel morphogenesis is critically dependent upon intercellular signaling and the regulation of gene expression. Although great progress has been made in the study of heart development, it is not known exactly how many genes and pathways are involved in this cardiogenic process or how many of these factors cooperate together. Previous genetic screens have identified genes that play roles in the specification, morphogenesis, and differentiation of the heart, including mastermind and tup. The current sensitized screen has also proved to be an efficient method to find additional factors in this process, suggesting that much remains to be learned about the molecular components involved in correct dorsal vessel formation (Xu, 2012).
Spir is required for the proper timing of cytoplasmic streaming in Drosophila, and loss of spir leads to premature microtubule-dependent fast cytoplasmic streaming during oogenesis, the loss of oocyte polarity, and female sterility. Even though it is known that spir is an important actin filament nucleation factor, the findings are the first report to describe a function of spir for cell division. Through phenotypic analysis of the spir mutant phenotype, it was found that many cardioblast nuclei are partially or completely divided. However, the cytoplasm is not divided in the absence of spir, which is consistent with the function of spir in cytoplasmic movement. Thirteen rapid nuclear division cycles without cell division initiate Drosophila embryo development, followed by three waves of cell division. The first wave of cell division occurs in the mesoderm at cell cycle 14. After this initial division, cells migrate, spread dorsally and undergo a second round of cell division at cell cycle 15. The third wave of cell division in the mesoderm occurs at the end of germband extension during cell cycle 16. There are two different types of cardioblast precursor cells: one type divides into two identical Tin-positive cardioblasts (TC), and the other type divides into one Svp-positive cardioblast (SC) and one Svp-positive pericardial cell (SPC). Based on the comparison of CycA and spir mutant phenotypes, a tentative cell division model is proposed to demonstrate spir function in determining cardiac cell fate (see A cell division model of spir function during heart development). In a wild-type background, one Svp-positive super progenitor (SSP) divides into two Svp-positive progenitors (SP), then each of these cells divides into one SPC and one SC. For Tin-positive super progenitors (TSP), after each divides into two Tin-positive progenitors (TP), each TP further divides into two identical TCs. In the current model, division from the super progenitor to progenitors takes place at cell cycle 15, and division from progenitors to full differentiated heart cells occurs at cell cycle 16. In CycA mutants, mitosis 16 is blocked such that both SPs and TPs stop cell division. This results in the two SPs assuming a myocardial fate. Thus the number of SCs remains normal, but half of the TCs are missing in the CycA mutants. The data suggest that in spir mutant embryos, most of the SPs fail to undergo full cell division at cycle 15 resulting in a SPC fate with paired nuclei. A subset of these cells are able to undergo the 15th cell division but are arrested at cycle 16 as double-nucleated cells which exhibit both Svp and Mef2 staining, characteristic of the SCs seen in the CycA mutants. Similarly, for TPs, cycle 16 was also blocked such that it resulted in two double-nucleated cells. In summary, Spir affects mitosis 16 for Tin-positive cell division and both mitosis 15 and 16 for Svp-positive cell division (Xu, 2012).
Antibody staining suggests that Spir is expressed ubiquitously before stages 12-13 and is located in both nuclei and cytoplasm. After cell cycle 16 cell division stops, occurring during stage 10-11. The expression of Spir in the cytoplasm then decreases gradually. At stage 15, the staining pattern shows mostly nucleus expression with some cytoplasmic expression and by stage 16 the nuclei become distinct indicating nucleus staining only. It is hypothesized that expression of Spir decreases in the cytoplasm but remains constant in the nuclei when cell division halts (Xu, 2012).
The genetic analysis of spir, Doc, pnr and tin suggests that these factors may regulate each other during dorsal vessel formation, and especially significant is the interaction between spir and pnr. Pnr is a GATA class transcription factor expressed in both the dorsal ectoderm and dorsal mesoderm, where it is required for cardiac cell specification. Proper dorsal vessel formation is inhibited in pnr loss-of-function embryos due to failure in the specification of the cardiac progenitors. In spir mutants, the expression pattern of Pnr remains normal. However, Spir is over-expressed in the cardiac mesoderm in pnr mutants, suggesting that Pnr may repress the expression of the spir (Xu, 2012).
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pannier:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 10 December 2020
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