tinman
Genetic
analyses indicate that tinman resides downstream of the mesodermal determinant twist, which encodes
a basic helix-loop-helix-type transcription factor. However, the regulation of tinman by twist remains
poorly understood. Using expression assays in cultured cells and transgenic flies, it has been shown that two
distinct clusters of E-box regulatory sequences, present upstream of the tinman gene, mediate tinman
expression in the visceral mesoderm. These elements are conserved between the Drosophila
melanogaster and Drosophila virilis tinman genes and serve as binding sites for Twist (E1 cluster located from -1134 to -1101) and
Tinman (E2 cluster located from -868 to -831) proteins. In cultured cells, Twist and Tinman binding results in the activation of tinman
gene expression. In transgenic animals, the E1 and E2 clusters are functionally connected; both
elements are required for tinman activation in cells of the visceral mesoderm and also for tinman
repression in cells of the somatic musculature. These results demonstrate that tinman is a direct
transcriptional target for Twist and its own gene product in visceral mesodermal cells, supporting the
idea that twist and tinman function in the subdivision of the mesoderm during Drosophila embryogenesis (Lee, 1997).
The Drosophila tinman homeobox gene has a major role in early mesoderm patterning: it determines
the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like
mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which
is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad
dorsal mesodermal domain, and finally restricted expression in heart progenitors. Each of these phases of expression is driven by a discrete enhancer element, the first being active in
the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. Surprisingly, all of these elements are located at positions downstream of the transcription start site. Element B(1800 bp) is located in the first intron; a second enhancer element, D (about 350bp), is located about 2 kb downstream of the 3' end of tin and activates gene expression in the dorsal portion of the mesoderm. Element D is active between stage 11 and early stage 12 of embryogenesis. A third element, C (300bp) is active in the dorsal vessel. This element activates expression from stage 12 on, in four out of six cardioblasts per hemisegment. Finally, an element A (about 500 bp), located in the 5' portion of the first intron, activates tin in the anterior tip of the head. After invagination of the stomodeum, the bulk of these tin expressing cells form the roof of the pharynx (Yin, 1997).
The early-active enhancer element is a direct target of twist, a gene necessary for tinman activation that encodes a basic
helix-loop-helix (bHLH) protein. This 180 bp enhancer
includes three E-box sequences that bind Twist protein in vitro and are essential for enhancer activity
in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal
cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. The 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes.
This repression requires the function of buttonhead, a head-patterning gene: buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the
same area. The second expression domain, restricting tin mRNA expression in the dorsal mesoderm, is triggered by Dpp-mediated induction events. Together, these results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restricts twist activity to specific areas within the twist expression domain (Yin, 1997).
The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like
portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with
the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior
subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B
(Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the
domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that
loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior
cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted
expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube
in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during
chamber specification in the developing vertebrate heart (Lo, 2002).
Antp is strongly expressed in four consecutive pairs of
cardioblasts in the anterior of the dorsal vessel.
The three anterior cardioblast pairs of this domain of strong
Antp expression are the posterior three tinman (tin) cardioblast pairs of segment A1, while the fourth pair corresponds to the
anterior pair of the two seven up (svp) cardioblast pairs located between A1 and A2. There is also strong expression in
at least six pericardial cells flanking the domain of strong
cardioblast expression, all of which are non-Tin expressing
pericardial cells. Weaker Antp expression is seen in a row of
three or four consecutive cardioblast pairs in T3 immediately
anterior to the domain of strong Antp, and also in the
four tin cardioblast pairs of segment A2 (Lo, 2002).
Since abd-A expression coincides with the heart portion
of the dorsal vessel, tests were made to see whether it acts to specify
the cardioblasts in which it is expressed to eventually form
the heart. In order to distinguish aorta cardioblasts from
heart cardioblasts, two different molecular markers were
utilized. The first marker was the pattern of ß-Gal derived
from the tinCdelta5-lacZ transgene, where the expression of a
lacZ gene is controlled by an internally deleted tinman
cardiac enhancer element, tinCdelta5.
This element drives ß-Gal expression in all the cardioblasts
of the aorta, whereas in the heart it is only expressed in
three segmentally-spaced double pairs of cardioblasts. These particular cardioblasts correspond to the
svp cardioblasts of the heart. The second marker
is wingless (wg), which is expressed in these same three
double pairs of svp cardioblasts within the heart of the late
embryonic dorsal vessel (Lo, 2002).
In abd-A null mutant embryos, the pattern of tinCdelta5-lacZ-derived ß-Gal is continuous in the heart as well as in
the aorta of the dorsal vessel. In addition,
it appears that the width of the heart is now the same as
that of the aorta when compared with a wildtype embryonic
dorsal vessel. Similarly, the late
expression of Wg in the svp cardioblasts of the heart is not
detectable in these mutant embryos. The alterations
in the pattern of these two markers strongly suggest
that heart cardioblasts have not been specified in the
posterior of the dorsal vessel of abd-A null mutant embryos
and that these posterior cardioblasts have been transformed
instead into aorta cardioblasts. This would indicate that
abd-A is necessary for the specification of heart cardioblasts
in the posterior portion of the dorsal vessel where it is
normally expressed (Lo, 2002).
tinman expression, except for a patch in the head, is dependent
on twist function. In contrast, snail, another mesoderm determinant does not appear to be
required for tinman initiation, but is necessary for the maintenance of tinman expression after
germband elongation (Bodmer, 1990).
After gastrulation, progenitor cells of the cardiac, visceral and body wall musculature arise at
defined positions within the mesodermal layer of the Drosophila embryo. An early and important event in the regional
subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells.
Genetic analysis has shown that this homeobox gene controls the formation of the visceral
musculature and the heart from dorsal portions of the mesoderm. An inductive
signal from dorsal ectodermal cells is required for activation of tinman in the underlying mesoderm.
Decapentaplegic serves as a signaling molecule in this process. The
spatial expression of dpp in the ectoderm determines which cells of the mesoderm become
competent to develop into visceral mesoderm and the heart (Frasch 1995).
The homeobox gene tinman plays a key role in the specification of Drosophila heart
progenitors and the visceral mesoderm of the midgut, both of which arise at defined
positions within dorsal areas of the mesoderm. In addition to the
heart and midgut visceral mesoderm, tinman is also required for the specification of all
dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral
musculature, and dorsal somatic muscles are all specified within the same broad domain
of dorsal mesodermal tinman expression. Locally restricted activities of tinman are also
observed during its early, general mesodermal expression, where tinman is required for
the activation of the homeobox gene buttonless in precursors of the dorsal median glial cells along the ventral midline. These observations, together with others
showing only mild effects of ectopic tinman expression on heart development, indicate
that tinman function is obligatory, but not sufficient to determine individual tissues
within the mesoderm. Therefore, it is proposed that tinman has a role in integrating
the positional information that is provided by intersecting domains of additional regulators
and signals, which may include Wingless, Sloppy Paired, and Hedgehog in the dorsal
mesoderm and EGF-signaling at the ventral midline. Previous studies have shown that
Dpp acts as an inductive signal from dorsal ectodermal cells to induce tinman expression
in the dorsal mesoderm, which, in turn, is needed for heart and visceral mesoderm
formation. In the present report, Thickveins, a type I receptor of Dpp, is
shown to be essential for the transmission of Dpp signals into the mesoderm. Constitutive activity of
Tkv in the entire mesoderm induces ectopic tinman expression in the ventral mesoderm,
and this results in the ectopic formation of heart precursors in a defined area of the
ventrolateral mesoderm (Yin, 1998).
Because of the crucial role of dpp in inducing dorsal mesodermal tinman expression and the specification of dorsal mesodermal tissues, it is of interest to determine whether other components known to function in dpp-mediated signaling events during blastoderm are also required for mesoderm induction. Screw, a second BMP2/4-related gene
product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein
Schnurri, are all shown to be required to allow full levels of tinman induction during this process. screw, which encodes a secod BMP2/4-related molecule, has been proposed to act synergistically with dpp to specify dorsal ectoderm and amnioserosa. Similarly, it has been shown that tolloid, which encodes a BMP1-related metalloproteinase, acts to enhance the activity of the dpp gene product during mesoderm induction. Both scw and tolloid are shown to be required for normal induction of tinman expression in the dorsal mesoderm, and in the absence of either gene activity, tinman expression in the dorsal mesoderm is reduced and segmentally interrupted. Thus scw and tld are necessary for achieving full levels of tinman induction, whereas dpp is obligatory for this event. In addition, unlike dpp mutants, mutants for scw or tld form some residual visceral mesoderm. However, heart formation is more sensitive to the activities of scw and tld and is disrupted to a similar extent as in dpp mutants. schnurri is also necessary for tinman induction in the dorsal mesoderm. The dorsal tinman domain is clearly reduced, as compared to wild-type embryos, although the levels of Tinman mRNA are close to normal. Therefore, shn may be required to enhance dpp signaling during tin induction, but significant levels of tin activation can still occur in the absence of its activity (Yin, 1998).
tinman is essential for dorsal vessel (heart) formation and is structurally and functionally conserved in vertebrates. In
the mature embryonic dorsal vessel, tinman is expressed in four of the six pairs of cardioblasts in each segment. Evidence that
seven-up, which is homologous to the vertebrate COUP-TF transcription factor and is expressed in the non-Tinman-expressing cardioblasts,
represses tinman in these cells. Loss of function seven-up mutations derepress tinman expression in these cardioblasts while ectopic
expression of seven-up represses tinman in the cardioblasts that normally express tinman. These changes are correlated with alterations in the
expression of additional molecular markers for each of these two types of cardioblasts, such as the novel T-box-containing gene Tb66F2 and
the potassium channel-encoding gene sur. These observations suggest that seven-up has a role in diversifying cardioblast identities within
each segment. The tinman cis sequences that mediate tinman repression by seven-up are described and whether Seven-up can
bind these sequences to directly inhibit tinman was examined. It seems that neither Svp isoform is capable of directly and
specifically binding to the 3' half of the tinC element that
confers segmental repression of tinman. It is still possible that Seven-up
could bind the tinC element in combination with one or
more other proteins in order to repress tinman, or Seven-up may indirectly repress tinman in svp cardioblasts through the regulation of downstream genes that affect tinman expression (Lo, 2001).
In late embryonic dorsal vessels, ß-Gal expression from ß-Gal is seen in two adjacent pairs of the six bilaterally symmetrical pairs of cardioblasts per segment. At these stages, Tinman is present in only four of
the six contiguous pairs of cardioblasts per segment, and
double-staining for ß-Gal and Tinman demonstrate that
the svp-lacZ-expressing cardioblasts correspond to the two
pairs of non-tinman-expressing cardioblasts. In order to extend this observation and better characterize seven-up expression in the entire dorsal vessel and its relationship to tinman expression, the pattern of ß-Gal and Tinman staining during the development of this
organ was examined in embryos from the svp-lacZ line
AE127. Tinman protein expression during early dorsal
vessel development (stage 11) includes all the heart progenitors, whereas in later stages it becomes restricted to the four pairs of cardioblasts per segment and to a subset of pericardical cells referred to as the tin pericardial cells. In AE127 embryos, the earliest detected expression of ß-
Gal in the Tinman-expressing heart progenitors is at mid-
stage 11 in a small subset of these cells, when it appears that
seven-up is simultaneously expressed in at least two heart
progenitor cells in each hemisegment. These cells are irregularly arranged and are first seen in the posterior half of the embryo. By early stage 12, a cluster of four strongly ß-Gal-expressing heart progenitors per hemisegment is seen in seven posterior segments of the embryo, with an additional pair of these cells situated caudally to these clusters. This pattern is maintained from stage 13 until stage 15, when strong ß-Gal expression is observed in two adjacent pairs of cardioblasts in seven segments, plus another single pair located immediately posterior to the last double pair of cardioblasts. The ß-Gal and Tinman double-staining of these late stage dorsal vessels clearly shows that the two pairs of seven-up-expressing cardioblasts correspond to the two pairs of cardioblasts per segment that are not expressing Tinman, such that the two patterns of expression are complementary. The late stage cardioblasts that exclusively express either tinman or svp-lacZ are referred to as the tin and svp cardioblasts, respectively. In addition to the svp cardioblasts, there is also strong expression of svp-lacZ at the anterior end of the dorsal vessel in two bilaterally symmetrical masses of cells that will later fuse at the dorsal midline to form the corpus allatum of the ring gland, an endocrine organ of complex origin (Lo, 2001).
Located laterally to each pair of svp cardioblasts per hemisegment is a pair of cells with much weaker expression of svp-lacZ, which do not express Tinman. Based on their lateral position relative to the cardioblasts and since they appear to arise from dividing svp-lacZ heart progenitors,
these cells are considered to be pericardial cells. Since they
do not express tinman, these are not tin pericardial cells and
thus constitute a novel subtype of pericardial cells. Prior to stage 13, all svp-expressing heart progenitors are
positive for Tinman protein (Lo, 2001).
Molecular characterization of the seven-up gene has identified two different transcripts, svp1 and
svp2, that are derived from this gene. The svp1 cDNA encodes a 543 amino acid protein that is the Drosophila homolog of the vertebrate COUP-TF subfamily of steroid/nuclear hormone receptors and
contains a characteristic N-terminal DNA-binding domain
and C-terminal ligand-binding domain. The svp2 cDNA
encodes a related 746 amino acid protein that is identical
in sequence to the svp1-encoded protein until it completely
diverges in the middle of the ligand-binding domain. Since
the pattern of ß-Gal expression in the AE127 svp-lacZ line
does not differentiate between these two transcripts and may
not reflect endogenous mRNA patterns in the heart and its
progenitors, in situ hybridizations were performed using
probes specific for the unique 3' UTR of each cDNA.
The svp1 in situ hybridization pattern in the dorsal vessel
during its development appears to be identical to the ß-Gal
expression observed in AE127 embryos. svp1 message is
first detected cytoplasmically in a portion of the heart
progenitors in stage 11 embryos, and in later stages the pattern of expression in the non-Tinman-expressing cardioblasts and the corpus allatum is
the same as observed with svp-lacZ.
However, while the pattern of dorsal vessel cells expressing
svp2 during embryogenesis appears to be identical to svp1,
the staining is not cytoplasmic but is instead concentrated in
one or occasionally two speckles per nucleus of those cells.
In a stage 11 embryo, the svp2 transcript is clearly seen as
speckles in the nuclei of a subset of heart progenitors. This speckled intranuclear localization is also seen in
several other tissues expressing the svp2 transcript, e.g. the
dorsal somatic muscles, but not in other tissues such as the
CNS, where it is cytoplasmic, indicating that the intracellular localization of the svp2 transcript is tissue-specific. In the dorsal vessel of stage 15 or older embryos, a pair of adjacent speckles is observed in
between the four tin cardioblasts of each hemisegment, with
each speckle associated with one svp cardioblast.
In addition, there is svp2 expression in the corpus allatum,
seen as two larger clusters of speckles at the anterior end of the dorsal vessel (Lo, 2001).
These studies have identified a novel function of seven-up in
determining one of two major cell fates in the cardioblasts, as
defined by expression of Tb66F2 versus tinman, through
repression of the alternative fate. As in the developing retina,
seven-up is expressed in a subset of cells in the dorsal vessel,
specifically, these cells are the double pairs of non-Tinman-expressing cardioblasts in each segment of the dorsal vessel.
The possibility that these two cardioblast types are functionally different in the mature embryonic heart is suggested first
by the exclusive expression in the tin cardioblasts of the sur
gene, which codes for the Drosophila homolog of the sulfonylurea receptor subunit of the vertebrate ATP-sensitive potassium ion channel. While the vertebrate sulfonylurea receptor has no intrinsic potassium ion channel activity, the Drosophila sur gene has additional sequences not present in the vertebrate SUR genes that endow it with this activity. The presence of the sur gene product in tin cardioblasts could conceivably result in a difference in the electrophysiological properties of these cardioblasts relative
to the svp cardioblasts, perhaps in the generation, propagation, and/or control of heartbeat in the dorsal vessel, since it is known that potassium ions are required for proper heartbeat function in Drosophila (Lo, 2001).
Two other differences of the svp cardioblasts from the tin
cardioblasts have previously been noted in a study of the embryonic dorsal vessel; the present study utilized as a marker the P-lacZ insertion line E2-3-9 that
has recently been identified as a svp-lacZ line. The first difference is that the svp cardioblasts are the cardioblasts initially contacted by the alary muscle cells; this is consistent with the alignment of these cells
with the alary muscles in mature embryonic dorsal vessels
as seen in the study. However, this morphological feature
is not visibly disrupted in svp mutant embryos. The
second difference is that in larval stages, these cardioblasts
still maintain their compact and rounded shape while the
other cardioblasts become larger and flattened. It has been
speculated that these svp cardioblasts may be involved in
the formation of the ostia -- segmentally repeated openings present in the larval and adult heart that have a valve-like function in allowing the inflow of
hemolymph into the heart during diastole. These differences
between the tin and svp cardioblasts suggest that the proper
specification of these two different cardioblast cell fates during embryogenesis may be crucial for correct dorsal vessel function (Lo, 2001).
In Drosophila, trunk visceral mesoderm, a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. These findings have been extended by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided in the A/P axis into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts a feedback effect on trachea and ectodermal derivatives (Hosono, 2003).
Metameric RNA expression of bnl, which encodes a ligand for
Breathless FGF receptor, is first observed as 12 patches at mid stage 11. bnl RNA
expression becomes homogeneous and then diminished during stage 12. tin is a homeobox gene that is required for dorsal mesodermal development. At early stage 10, tin is expressed throughout the
dorsal mesoderm from which VM is derived. Metameric Tin expression becomes
evident by early stage 11. Tin
expression decreases during stage 12. Expression of bap, another
homeobox gene required for VM development, can be monitored by bap
4.5#230; (bap-lacZ). Staining for Tin and bap-lacZ or bnl RNA
indicates that
tin, bap and bnl are co-expressed in VM-PS3-12 during stage
11; in VM-PS2, only bnl is expressed. Stage 11-12 VM also
stains for Tin and VM-hh-lacZ. Tin and
VM-hh-lacZ expression partially overlaps. VM-hh expression
in the anterior terminal region of VM-PSs indicates that each
tin/bnl/bap trio expression domain straddles the VM-PS boundary (Hosono, 2003).
In summary, VM-PSs in thorax and abdomen, respectively, are
subdivided into five or six regions with respect to differential expression of
VM-metameric genes at stages 11-12. Detailed analysis of VM-hh, bnl, tin
and bap expression in addition to con indicates that trunk
visceral mesodermal genes are classified into three distinct groups --
tin/bnl/bap, VM-hh and con -- and each VM-PS
is subdivided into five or six regions, which become apparent during mid stage 11
to stage 12 (Hosono, 2003).
VM is presently considered to develop in two steps under the control of
ectodermal Hh and Wg signals. First, by stage 10 (when four mesodermal
primordia have become specified), VM competent or bap expression
regions are promoted by hh but repressed by wg, via a direct
targetor, slp. The second surge of hh and wg activity at
stages 10-11 is responsible for subdividing VM-PSs into two regions:
con positive and negative. These results indicate that the expression of
four other VM-metameric genes, hh, tin, bnl and bap, is also
regulated by the second surge of hh and wg activity at
stages 10-11 (Hosono, 2003).
In view of morphological changes in a VM competent region and consideration
of these findings on VM gene regulation, the following model for VM-PS cell
specification is proposed. At stage 10 to early stage 11, anterior terminal cells of VM-PSs are
presumed to be situated near an ectodermal AP border, where they are
capable of continuously receiving Wg and Hh signals, and Wg confers competence
on these cells to express tin/bnl/bap. Wg and Hh are responsible for
inducing VM-hh, and Hh, for con expression. In the
anterior-most cells, con expression is reduced, which would be
expected in view of repression by high Wg signal. The different thresholds of
hh for con and VM-hh expression may explain why the
con area expands more posteriorly compared with that of
VM-hh. Posterior terminal VM cells, when formed, are situated far
from Wg expressed on the ectodermal PS border. But as they migrate
posteriorly and close to the posteriorly neighboring AP border by early stage
11, they become capable of receiving Wg and acquire competence to express
tin/bnl/bap. Thus, the
tin/bnl/bap domain would appear regulated by spatially and temporally
distinct Wg signals. The two-step induction of tin/bnl/bap expression
is supported by experiments using the wgts mutant, where,
either posterior or anterior expression within one patch can be
differentially turned off. Indeed, a stepwise activation of
tin/bnl expression is seen in VM-PSs around stage 11. tin
and bnl metameric expression became apparent almost simultaneously at
mid-stage 11, and preliminary experiments have shown that neither tin
nor bnl misexpression can induce the ectopic expression of any
other metameric genes examined here. Thus, tin and bnl
expression might be initiated in a mutually independent manner (Hosono, 2003).
Inductive signaling is of pivotal importance 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, 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).
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).
This study showS that cell fate decisions in the dorsal and lateral mesoderm of Drosophila depend on the antagonistic action of the Gli-like transcription factor Lame duck (Lmd) and the zinc finger homeodomain factor Zfh1. Lmd expression leads to the reduction of Zfh1 positive cell types, thereby restricting the number of Odd-skipped (Odd) positive and Tinman (Tin) positive pericardial cells in the dorsal mesoderm. In more lateral regions, ectopic activation of Zfh1 or loss of Lmd leads to an excess of adult muscle precursor (AMP) like cells. It was also observed that Lmd is co-expressed with Tin in the early dorsal mesoderm and leads to a reduction of Tin expression in cells destined to become dorsal fusion competent myoblasts (FCMs). In the absence of Lmd function, these cells remain Tin positive and develop as Tin positive pericardial cells although they do not express Zfh1. Further, it was shown that Tin repression and pericardial restriction in the dorsal mesoderm facilitated by Lmd is instructed by a late Decapentaplegic (Dpp) signal that is abolished in embryos carrying the disk region mutation dppd6 (Sellin, 2009).
Loss of Lame duck (Lmd) leads to an increase of pericardial cells and adult muscle precursor like cells: In embryos lacking Lmd function, staining for zinc finger homeodomain factor 1 (Zfh1) expression reveals a pericardial hyperplasia phenotype and a general excess of Zfh1 positive mesodermal cells. In wild type embryos, three types of pericardial cells (PCs) have been described: Tin positive (TPCs), Odd positive (OPCs) and Eve positive (EPCs) pericardial cells, all of which express Zfh1 and the handC- GFP reporter. Closer inspection of the pericardial cells in lmd mutant embryos revealed that the number of TPCs and OPCs is dramatically increased, while the number of EPCs is normal. All OPCs co-express the handC- GFP reporter and Zfh1 in wild type and lmd mutant embryos. In contrast, a considerable number of ectopic Tin positive cells, though positive for handC- GFP, do not express Zfh1 in lmd mutant embryos. The absence of β3Tubulin expression in these cells is consistent with earlier reports in which a normal set of cardioblasts was described in lmd mutant embryos. To decide whether the Zfh1 negative/Tin positive cells are atypical pericardial cells or dorsal mesodermal cells that fail to differentiate, a triple staining waa conducted for Tin, Zfh1 and Pericardin (Prc), a collagen that is secreted by differentiated pericardial cells. Prc protein was observed surrounding all Tin positive/Zfh1 negative cells, suggesting that they are ectopic pericardial cells. However, due to the fact that Prc is a secreted protein the possibility cannot be ruled out that there might be occasional Tin positive/Zfh1 negative cells in lmd mutant embryos which do not express Prc themselves, but remain in an uncommitted, dorsal mesodermal state (Sellin, 2009).
For further analysis of the ectopic pericardial cells, the number of OPCs in stage 16-17 embryos was counted. An average of 206.3 OPCs was observed in lmd mutant embryos as compared to 97.8 in wild type embryos, thereby representing a ~2-fold increase. It has been reported that the Odd subgroup of pericardial cells (OPCs) originates from two different lineages: a symmetric lineage (two OPCs from one precursor) and an asymmetric lineage (two OPCs from two precursors), adding up to a total of four OPCs per hemisegment. Of note, the siblings of the asymmetrically derived OPCs, the Seven-up (Svp) positive cardioblasts, are normal in lmd mutants, thus suggesting that the asymmetrically derived OPCs do not contribute to the lmd phenotype. Since the two different types of OPCs can not be distinguished directly because the anti-Svp antibody stains the precursor cells and the cardioblast siblings, but not the final PCs at later stages, their abundance was measured in lmd mutants indirectly. The fact was utilized that in inscutable (insc) mutants, asymmetric cell division fails, and all siblings of the asymmetric OPC lineage become Svp positive cardioblasts. The difference in OPC number between insc; lmd and lmd mutant embryos therefore corresponds to the number of asymmetrically derived OPCs in lmd mutant embryos. A loss of ~45 OPCs was observed in insc; lmd double mutant embryos as compared to lmd mutant embryos. This number is reasonably close to the number of ~38 OPCs that are lost in insc mutant embryos when compared to wild type embryos. In addition, the number of Svp positive precursors, which give rise to the asymmetric Odd lineage, is normal in lmd mutant embryos at early stage 13. Altogether, these data strongly support the initial hypothesis that there is the normal amount of asymmetrically derived OPCs in lmd mutant embryos and the phenotype is not caused by a failure of asymmetric cell division (Sellin, 2009).
An excess of Zfh1 positive cells was also observed in the lateral mesoderm of lmd mutant embryos, where it is normally expressed in the adult muscle precursor cells (AMPs). These imaginal myogenic cells retain Twist (Twi) expression, but do not express any other myogenic genes in the embryo. Instead, they are maintained in a less differentiated state during embryogenesis and are dormant until metamorphosis, when they start to differentiate and give rise to the adult musculature of the fly. In the embryo, they are arranged as groups of cells in the thoracic segments, while six solitary cells (one dorsal, two dorsolateral, two lateral and one ventral) are present in the abdominal hemisegments. It was reported earlier that too many Twi positive cells persist in the lateral mesoderm of lmd mutant embryos. Together with the fact that both Zfh1 and Twi are present in AMPs in the wild type, it appeared likely that both factors are also co-localized in embryos mutant for lmd. Indeed, double staining for Zfh1 and Twi showed a complete overlap in the lateral mesoderm and confirmed that both populations of ectopic cells are identical. They also express the gene holes in muscles (him) which is another marker specific for AMPs. For further characterization, the expression patterns were analyzed of several myogenic markers in lmd mutant embryos. No expression was detected of the muscle specific genes myocyte enhancing factor 2 (Mef2), β3 Tubulin or the reporter rP298 (Duf-lacZ) in Twi/Zfh1/Him positive cells (Sellin, 2009).
Twist expression is normally present during early stages of somatic muscle development in myoblasts that are not yet differentiated. Zfh1, which has been implicated in the repression of mef2, might help in keeping AMPs in the undifferentiated state until metamorphosis. The gene him, which is also expressed in AMPs, was recently reported to be involved in maintaining cells in an undifferentiated state by inhibiting the myogenic signal provided by Mef2 function. Consequently, the ectopic Zfh1/Twist/Him positive cells in the lmd mutant embryos are likely to be cells with myogenic potential, as are the endogenous AMPs, and hence can be considered to be ectopic AMP like cells. To assess if enhanced proliferation is also involved in generating an increased amount of cells in lmd mutant embryos, staining was oerfirned for phosphorylated Histone 3 (pH3), which specifically marks dividing cells. Over-proliferation in the dorsal and lateral mesoderm was not observed in lmd mutant embryos when compared to wild type embryos. Although there is a considerable number of the additional, AMP like cells that persist until the end of embryogenesis, their number is reduced between stage 13 and 16/17. Staining with Nile Blue A revealed a general excess of dying cells during these stages in lmd mutant embryos as compared to wild type, suggesting that not all ectopic cells survive until the end of embryogenesis (Sellin, 2009).
The supernumerary PCs and AMPs originate from the population of fusion competent myoblasts: While no general myogenic genes are expressed in the ectopic AMP like cells, it was however possible to show co-localization of Zfh1 and lmd mRNA in the somatic mesoderm of lmd mutant embryos, which is not observed in wild type embryos. In the wild type, lmd is expressed in fusion competent myoblasts (FCMs), which fail to differentiate in the absence of Lmd function. lmd mRNA is transcribed in a normal pattern in lmd mutant embryos. In situ hybridization with a lmd specific riboprobe therefore allowed visualization of the population of cells destined to become FCMs, although they do not express any other FCM specific genes in lmd mutant embryos. Since the ectopic Zfh1 positive cells co-express lmd mRNA, it is concluded that the ectopic AMP like cells in lmd mutant embryos originate from the FCM population and adopt AMP like characteristics instead. They are therefore generated by cell fate conversions, which is consistent with the observation that there are no additional cell divisions in lmd mutant embryos (Sellin, 2009).
In the dorsal mesoderm of lmd mutant embryos, the additional Zfh1 positive cells express Tin or Odd and Prc, indicating differentiation as pericardial cells. Pericardial cells usually develop from the dorsal cardiac mesoderm specified by Tin expression, while the somatic musculature is situated more laterally and is characterized by prolonged Twi expression. To address the question of whether a conversion of FCMs into PCs could also account for the pericardial hyperplasia phenotype in lmd mutant embryos in an analogous fashion to the ectopic AMP like cells, staining was carried out for Tin and lmd mRNA. It was reasoned that the ectopic Tin cells should also express lmd mRNA if they originate from the pool of mis-specified FCMs. Indeed, there is co-expression of lmd mRNA and Tin in ectopic pericardial cells in stage 13 lmd mutant embryos, indicating that cell fate conversions from FCM to ectopic PC fate are responsible for the observed pericardial hyperplasia phenotype (Sellin, 2009).
Of note, there is a distinct overlap of lmd mRNA and Tin expression in the dorsal mesoderm of stage 12 embryos, both in wild type and lmd mutant background. This observation is consistent with the observed cell fate switch from FCM to PC fate and indicates that in wild type embryos, dorsal FCMs are specified in the dorsal, Tin positive mesoderm rather than the Twi positive somatic mesoderm. Indeed, dorsal muscle phenotypes can be observed in embryos mutant for tin, consistent with the conclusion that dorsal muscle cell types (i.e., FCMs) develop from the early dorsal mesoderm specified by Tin expression: If this domain is not specified, it can not generate dorsal FCMs (or other dorsal mesodermal derivatives, like heart or visceral mesoderm) (Sellin, 2009).
Co-expression of Tin and lmd mRNA is no longer detectable after germ band retraction (stage 13) in wild type embryos, but persists in lmd mutant embryos until the lmd mRNA signal fades (at about stage 14-15). Thus, it seems that repression of Tin in the dorsal mesoderm depends on the presence of Lmd protein. To substantiate this observation, Lmd was overexpressed in the whole mesoderm with the twi-Gal4 driver to assess its influence on Tin expression. Indeed, a reduction was observed of Tin expression in stage 12 embryos overexpressing Lmd compared to wild type, further confirming a negative influence of Lmd on Tin expression in the dorsal mesoderm. At later stages, the number of TPCs (and OPCs) remains reduced, while the cardioblasts are not affected. A model is therefore proposed in which the initial dorsal mesoderm specified by Tin expression is subdivided by Lmd into cardiac mesoderm and dorsal musculature by repression of Tin in lateral regions and induction of a myogenic differentiation program instead. During this process, Tin expression is maintained only in the cells that are destined to become pericardial cells (or cardioblasts), while Tin is repressed by Lmd in the dorsally localized FCMs. Loss of Lmd function consistently leads to an increased amount of Tin positive cells in the dorsal mesoderm from stage 13 onwards, which then can differentiate as ectopic pericardial cells as indicated by the expression of Prc. Taken altogether, these data suggest that, in the absence of Lmd function, the pool of unspecified FCMs can develop as ectopic PCs in the Tin-positive dorsal mesoderm and as AMP-like cells in the lateral and ventral mesoderm. However, increased cell death, and the possibility that a small number of ectopic Tin positive cells might exist without Prc/Zfh1 expression as mentioned earlier, suggest the possibility that not all cells of the FCM population follow alternative cell fates. Instead, some cells might remain in an uncommitted mesodermal state in lmd mutant embryos (Sellin, 2009).
Normally, instructive Dpp signals from the ectoderm are responsible for the specification of cardiac cell types by maintaining Tin expression solely in the dorsal mesoderm, while Twist activity in the lateral and ventral mesoderm leads to the development of the somatic musculature. To test if reduced Dpp signaling has a similar effect on PC number as overexpression of Lmd, by reducing the size of the Tin domain, embryos carrying the mutation mad1-2 were examined. mad1-2 is a weak hypomorphic allele of the Dpp effector Mad and causes larval lethality, thereby allowing observation of late stages of embryogenesis. Indeed, a decreased number of OPCs and TPCs was observed in mad1-2 mutant embryos, without any effect on cardioblast number, as is the case when overexpressing Lmd. Of note, the number of OPCs is decreased to a similar extent in mad1-2; lmd double, as compared to lmd single mutant embryos. Therefore, it is concluded that in the presence of the hypomorphic mad1-2 mutation, the dorsal mesoderm that is specified by Dpp-dependent Tin expression is reduced, resulting in a reduction of PCs in a Lmd independent manner. However, Lmd further restricts the number of PCs in the mad1-2 mutant background, as revealed by an increased number of PCs and the presence of TPCs without Zfh1 expression in mad1-2; lmd double mutants when compared to mad1-2 single mutants (Sellin, 2009).
Pericardial cells share their developmental origin with the myogenic cardioblasts in a similar fashion as AMPs with founder cells in the somatic musculature. During lateral inhibition, Notch activation promotes myogenic FCM fate as opposed to the progenitors of founder cells in the lateral mesoderm or cardiogenic progenitors in the dorsal mesoderm. Subsequently, during the process of asymmetric cell division, Notch activation renders the daughter cell always non-myogenic (PC or AMP fate). Although the AMPs have the potential to develop into muscle cells during metamorphosis, they are considered non-myogenic in this context because they do not yet express any myogenic genes, such as mef2, lmd or muscle structural genes in the embryo. In the case of pericardial cells, there is surprisingly little data available about their physiological role. While it is known that the OPCs contribute to the population of nephrocytes in postembryonic stages, TPCs and EPCs are not correlated with any function at all, and their developmental fate after embryogenesis is still unknown. A recent study described the development of adult muscular structures, the so called wing hearts, from a specialized subset of EPCs. This is the first hint that some pericardial cells might be considered as imaginal myogenic cells in an analogous fashion to AMPs, and it highlights the necessity to further characterize pericardial cells (Sellin, 2009).
It is currently known that PCs and AMPs have in common a dependency on active Notch signaling although they stem from different cell lineages and mesodermal primordia (Tin vs. Twi domain). FCMs, which adopt AMP or PC like characteristics in lmd mutant embryos, also need active Notch signaling. In fact, Lmd is a downstream target of N signaling and induces the FCM differentiation program. The observed lmd phenotype could be explained if, in the absence of Lmd, Notch activity always promoted AMP or PC (non-myogenic) fate, but not FCM fate, independently of the original pathway that is involved (lateral inhibition or asymmetric cell division). To assess this hypothesis, double mutants for lmd and genes involved in the Notch pathway were established. For this analysis kuzbanian and mastermind alleles were chosen because loss of either gene causes lethality only late in embryogenesis due to a maternal component, thereby allowing the analysis of later events in heart and muscle development. Both genes have also been well studied with respect to their molecular function and developmental implications. Kuzbanian (Kuz) is an ADAM metalloprotease that is known to process the Notch receptor following ligand binding. Zygotic loss of function mutations lead to defects in both lateral inhibition and asymmetric cell division in heart and muscle development, although the phenotype is far weaker than in embryos carrying N loss of function alleles. mastermind (mam) is involved in transducing the Notch signal and displays a stronger heart phenotype than kuz and a mild Notch-like muscle phenotype. Staining was perfomed for expression of Krüppel (Kr) and him mRNA, which are specific for a subset of muscle founders and AMPs/ PCs, respectively, and an increase of Notch negative cell types, corresponding to founders, was observed in the somatic mesoderm of kuz mutant embryos. This is accompanied by a reduction of AMPs, confirming the expected function of Kuz in facilitating N function in muscle cell differentiation. Furthermore, the number of FCMs as marked by Lmd expression is strongly reduced in kuz mutants, although the effect is not as complete as in N loss of function alleles (Sellin, 2009).
In kuz; lmd double mutant embryos, the increase of AMPs is milder than in lmd mutant embryos, which is consistent with a failure in lateral inhibition and a concomitant reduction of FCMs that are available for conversion to AMPs. The number of Kr-positive founder cells is increased to comparable levels in kuz and kuz; lmd mutant embryos, suggesting that Notch inactive cell fates (muscle founders and cardioblasts) are not influenced by the absence of Lmd, and that Notch acts as a permissive signal to allow the cell fate switch in lmd mutant embryos. mam; lmd double mutant embryos display a similar phenotype. Altogether, these findings suggest that in the double mutants, a general reduction of cell types with Notch activity (i.e. FCMs) occurs, followed by the conversion of the remaining potential FCMs to AMP or PC fate under the influence of N signaling in the absence of Lmd. Lame duck is present in stages 12-14, which is later than the period during which Notch activity is involved in facilitating cell fate decisions within the musculature. Hence, it appears that Notch can promote AMP or PC fate at a relatively late time point in the absence of Lmd (Sellin, 2009).
It was of interest to know if the endogenous set of AMPs, which develop through asymmetric cell divisions of muscle progenitors, is specified correctly in lmd mutant embryos. For example, the lateral AMPs are the siblings of the segment border muscle founder (SBM), and share with the latter the expression of the identity factor Ladybird early (Lbe). To discern ectopic cells and endogenous AMPs in lmd mutant embryos, co-staining was performed for Lbe and Twi expression. Indeed, the normal number of lateral AMPs, as marked by Lbe expression, is present in lmd mutant embryos, while far too many Twi-positive cells was observed in general. The latter are the ectopic AMP like cells that are presumed to be recruited from the FCM population. This observation further confirms that individual mesodermal lineages, such as the asymmetrically derived OPCs or individual AMPs, are not influenced by the loss of Lmd function (Sellin, 2009).
The proposed model of cell fate switches from myogenic FCM fate to non-myogenic AMP like or PC fate, but not myogenic fates (cardioblasts or founder cells), is consistent with the observation that Notch signaling is often employed to delay or inhibit the differentiation of stem cells or progenitor cells, especially in myogenesis. In vertebrates, Notch signaling prevents satellite cells (muscle stem cells) from entering a myogenic differentiation program in cell culture as well as in vivo, and impaired upregulation of its ligand Delta-like 1 in satellite cells has been correlated with a decreased capacity of aging muscle tissue to regenerate. While the data are consistent with the general function of Notch in preventing cells to enter the myogenic differentiation program by promoting the AMP or PC fate, they also highlight the special and unusual properties of Lmd - as a target of Notch signaling - in Drosophila muscle development. Although it is activated by Notch, it has the ability to induce myogenic differentiation. The data strongly suggest that the AMP or PC fate is the default consequence of Notch signaling in Drosophila myogenesis and that Lmd function overrules this signal to induce the FCM differentiation program in lateral or dorsal competence domains. It was shown that N has a biphasic function in heart differentiation analogous to the situation in the somatic mesoderm. At an early phase, N activity restricts the number of the sum of CBs and PCs, reflecting a function in the definition of early cardiac progenitors, likely by lateral inhibition. Subsequently, N activity is needed to promote pericardial cell fates in asymmetric cell division of the early progenitors. Although the last division step is in many cases a symmetric division seem to indicate that the majority of cardiac cell types is generated by asymmetric cell divisions segregating cardiac and pericardial fates. This might occur in some cases at one of the earlier division steps of the progenitor(s). Since these data indicate the generation of FCMs from the dorsal mesoderm, as reflected by co-expression of Tin and Lmd in stage 12 embyos, it might be suggested that dorsal FCMs originate from dorsal competence domains which also give rise to the above mentioned cardiac progenitors. These progenitors divide asymmetrically to generate CBs and PCs analogous to FC/AMP sibling pairs from more lateral competence fields, while it ia proposes that all or some of the remaining cells of the competence domains begin to express Lmd and generate FCMs under instructive influence of N signaling. In the absence of Lmd function (either in wild type in the N active daughter cells of the progenitors, or in lmd mutant embryos in all N active cells of the competence domains), the N signal promotes non-myogenic cell fates according to the mesodermal context (i.e., dorsal vs. lateral mesoderm). This would then result in the differentiation of the non-segregating population (normally developing as FCMs) as PCs in the Tin domain and AMPs in the somatic mesoderm (Sellin, 2009).
Lame duck and Zfh1 act antagonistically in mesodermal cell fate decisions: While loss of Lmd function results in an increased number of Zfh1-positive cell types, overexpression of Lmd leads to the opposite phenotype. The pan-mesodermally active twi-Gal4 driver line was used to induce Lmd expression in the whole mesoderm, and a reduction of OPCs, TPCs and AMPs was observed. To assess whether pericardial cell reduction might be a secondary effect of the early Tin repression caused by ectopic Lmd activity, the later and cardiac specific handCA-Gal4 driver, which is active in the heart from stage 12 onwards, was used. At this time point, the OPC precursors are already specified and are no longer expressing Tin. Since hand>Lmd overexpression severely reduces the number of all pericardial cells, it is concluded that their reduction is not only a secondary effect of the narrower Tin domain in embryos overexpressing Lmd. To further confirm this conclusion, the phenotype of zfh1 mutant embryos was compared with that of embryos overexpressing Lmd. The number of OPCs and TPCs is also reduced in zfh1 mutant embryos quite similarly to embryos overexpressing Lmd, although the early Tin expression pattern is normal in the absence of Zfh1 function. It is therefore unlikely that Lmd acts negatively on Zfh1 expression only by reducing Tin expression, but rather also independently of Tin function (Sellin, 2009).
There are however important differences in the phenotypes of twi > Lmd and zfh1 mutant embryos. Zfh1 appears to be involved in maintaining, but not in specification of OPCs, because it has been observed that loss of Zfh1 does not affect the number of OPC precursors at stage 13, but rather leads to a decrease of OPCs at later stages. This is in contrast to a reduced number of OPC precursors in stage 13 embryos overexpressing Lmd. Therefore, Zfh1 repression alone can not account for the loss of PCs in embryos ectopically expressing Lmd. Instead, it might be that the reduction of the dorsal Tin domain by ectopic Lmd expression results in the specification of fewer OPC precursor cells, followed by further reduction of the remaining OPCs by the negative effect of ectopic Lmd on Zfh1 expression. Consistently, a much stronger reduction of OPCs was observed after ectopic expression of Lmd as compared to the loss of OPCs in zfh1 mutant embryos. The observation that loss of Lmd function leads to the appearance of TPCs that do not express Zfh1, but Prc as a marker of pericardial differentiation, is another hint that both effects occur independently of each other and that pericardial differentiation can be accomplished in the absence of Zfh1 in lmd mutant embryos (Sellin, 2009).
Taken altogether, it does not seem likely that Tin and Zfh1 act in an epistatic hierarchy in dorsal mesodermal cell fate decisions. Instead, the data support the conclusion that Lmd regulates OPC and TPC number by two independent mechanisms: (1) Initially, Lmd restricts the cardiac field in general through repression of Tin, which leads to the reduction of early OPC precursors and the elimination of Tin expression in cells that do not express Zfh1 (which can differentiate as TPCs, as indicated by Prc expression, in the absence of Lmd function). (2) Later, it represses Zfh1, thereby reducing further the number of OPCs and TPCs. This is consistent with previous findings which described Zfh1-dependent and Zfh1-independent mechanisms for the regulation of OPC and TPC number (Sellin, 2009).
Of note, it was previously shown that Zfh1 overexpression leads to an increase in pericardial cell number (both OPCs and TPCs) and a concomitant loss of dorsal somatic muscle cells, indicating that overexpression of Zfh1 phenocopies the pericardial hyperplasia in lmd mutant embryos. It was shown further that overexpression of Zfh1 with the twist-Gal4 or 24B-Gal4 driver leads to an increased number of AMP like cells in the dorsal mesoderm although the effect is rather weak when compared to lmd mutant embryos. Zfh1 overexpression does not however alter the pattern of Lmd expression, indicating that Zfh1 does not antagonize Lmd function at the transcriptional level. To verify whether Zfh1 has an influence on Lmd at the posttranscriptional level, the intracellular distribution of Lmd was analyzed in embryos overexpressing Zfh1, because Lmd function has been shown to be modulated by its subcellular localization in wild type embryos. In embryos overexpressing Zfh1, the subcellular localization of Lmd does not appear to be altered, suggesting that Zfh1 does not influence the subcellular distribution of the Lmd protein (Sellin, 2009).
Taken together, these data indicate that Lmd and Zfh1 have generally opposite effects on dorsal mesoderm differentiation: Lmd loss-of-function or Zfh1 gain-of-function leads to increased AMPs or PCs, whereas Lmd gain-of-function and Zfh1 loss-of-function reduce these cell types. Consequently, Lmd and Zfh1 can be considered to be functional antagonists, although their repression is not mutual. One possible explanation for the antagonistic effect of Zfh1 overexpression might be due to its direct negative influence on mef2 expression, thereby counteracting the mef2 activating function of Lmd. The vertebrate functional orthologue of Zfh1, ZEB2 (or Sip1), also inhibits myotube development in culture and represses a number of myogenic genes, and is able to rescue Zfh1 function in Drosophila (Sellin, 2009).
Lmd is instructed to restrict Tin expression by a late, pro-myogenic Dpp signal: While in wild type embryos Tin expression is repressed in cells destined to become dorsal FCMs between stages 12 and 13, there is a prolonged co-localization of Tin and lmd mRNA in cells of the dorsal mesoderm in lmd mutant embryos. As a consequence, dorsal FCMs adopt pericardial cell fates in the absence of Lmd function. Of note, this effect can also be observed in embryos carrying the dppd6 disk region mutation. These embryos lack a late Dpp signal (beginning at about stage 12) that is involved in pericardial restriction. Early Dpp signaling does not seem to be affected since the dorsal mesoderm (characterized by Dpp-dependent Tin expression) is normal in dppd6 mutant embryos. Quite contrary to embryos with otherwise decreased Dpp signaling and a reduced pericardial field, such as mad1-2 embryos, the dppd6 mutant embryos display a pericardial hyperplasia phenotype that resembles in many aspects the phenotype observed in lmd mutant embryos. Too many OPCs, TPCs and atypical TPCs without Zfh1 expression are also detected, although the dppd6 mutant phenotype is milder than the lmd mutant phenotype. This resemblance in phenotypes suggested an epistatic relationship of Lmd and the late Dpp signal. In addition, the accumulation of phosphorylated Mad (pMad) has been traced in PCs and cells within the dorsal musculature that are not positive for founder specific Kr or Eve expression, and hence are likely to be FCMs. Altogether, these findings lead to the hypothesis that Lmd might be a target of the late Dpp signal in FCMs. However, Lmd is expressed in a normal pattern (both at the mRNA and protein levels) in dppd6 mutant embryos, indicating that Lmd expression is independent of Dpp signaling. Nevertheless, co-staining with anti-Tin antibody revealed a prolonged co-localization of Tin and lmd mRNA in dppd6 mutant embryos until stage 14/15, as observed in lmd mutant embryos, suggesting a requirement for late Dpp signaling in the process of pericardial restriction by Lmd. To assess if the restrictive influence of late Dpp signaling on Tin expression is indeed relayed by Lmd in the dorsal mesoderm, or if both negative effects are independent of each other, the late Dpp signal was enhanced in the lmd mutant background. For this purpose, the leading edge driver LE-Gal4 was used to overexpress Dpp, which was shown to reduce the number of OPCs and TPCs in the wild type background. It was reasoned that this effect would be lost in lmd mutant embryos if Lmd is responsible for the restricting effect on PC number. The number of OPCs was counted in LE > Dpp; lmd embryos in comparison to lmd mutant embryos. While overexpression of Dpp with the LE-Gal4 driver in the wild type background led to a reduction of OPCs by ~1.2-fold, no reduction of OPCs was observed in the lmd mutant background, indicating that Lmd is indeed necessary to interpret the late Dpp signal as pro-myogenic. Altogether, these data suggest that the pro-myogenic effect of the late Dpp signal is Lmd dependent, although not by inducing Lmd expression. Instead, the presence of Dpp activity seems to be a prerequisite for the negative influence of Lmd on Tin expression and might act as an instructive signal to modify Lmd activity to allow repression of Tin. If the late Dpp signal is lost -- as is the case in embryos carrying the hypomorphic allele dppd6 - repression of Tin fails even in the presence of Lmd protein, indicating that repressive activity of Lmd is dependent on Dpp signaling (Sellin, 2009).
A model is proposed in which the subdivision of the early Tin positive primordium into pericardial and dorsal muscle tissues is mediated via the antagonistic action of Lmd and Zfh1 under the instructive influence of late Dpp signals. While the early function of Dpp restricts Tin expression to the dorsal mesoderm, subsequent Dpp signaling provides pro-myogenic input to modulate the pericardial field in favor of the dorsal musculature. The present data show that the function of this late Dpp signal requires Lmd activity, strongly suggesting that Lmd is a target of Dpp for establishing the boundary between the dorsal musculature and pericardial field. Repression of Tin also appears to be dependent on Dpp signaling. The previous observation that pMad accumulation occurs in PCs and dorsal muscle cells, which are likely to be FCMs, is consistent with the finding that Lmd is needed to relay the pro-myogenic function of late Dpp signaling. These cells originate from the Tin-expressing dorsal mesoderm, and co-expression of Tin and lmd mRNA in wild type embryos at stage 12 can be observed. In the presence of Lmd protein, this co-expression is not maintained after stage 12 due to a repressive function of Lmd on Tin. Of note, it was previously shown that Lmd function depends on posttranscriptional mechanisms that modulate its specific subcellular localization and activity, and it might be speculated that Dpp signaling is involved in changing Lmd function into a repressive form. However, there is no evidence that the negative influence of Lmd on Tin expression is of a direct nature, or if there are other factors that are involved in the process. In this context, the following explanation for the antagonistic effect of Zfh1 overexpression without repression of Lmd could also be considered. Since the vertebrate homologue ZEB2 was shown to inhibit activation of target genes by Smads, an excess of Zfh1 might antagonize the late Dpp signal by repressing pMad-dependent interaction partners of Lmd, thereby preventing the repression of Tin (and/or other targets) in the dorsal mesoderm. Lmd expression and function would not be affected elsewhere which would be consistent with the observation that Zfh1 is not a general repressor of Lmd (Sellin, 2009).
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