tinman
bagpipe, found downstream of tinman, is involved in the differentiation of ventral visceral mesoderm (gut musculature). While the defect in ventral visceral mesoderm invagination is partial in tinman mutants, it is absolute in bagpipe mutants (Azpiazu, 1993).
The function of the Drosophila mef2 gene, a member of the MADS box supergene family of
transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely
somatic, visceral, and cardiac. During embryogenesis, mef2 undergoes multiple phases of expression,
which are characterized by initial broad mesodermal expression, followed by restricted expression in
the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the
differentiated musculatures. Evidence is presented that temporally and spatially specific
mef2 expression is controlled by a complex array of cis-acting regulatory modules that are responsive
to different genetic signals. Functional testing of approximately 12 kb of 5' flanking region of the mef2
gene shows that the initial widespread mesodermal expression is achieved through a 280-bp
twist-dependent enhancer. Subsequent dorsal mesoderm-restricted mef2 expression is mediated
through a 460-bp dpp-responsive regulatory module, which involves the function of the Smad4 homolog
Medea and contains several binding sites for Medea and Mad. Regulated
mef2 expression in the caudal and trunk visceral mesoderm, which give rise to longitudinal and circular
gut musculatures, respectively, is under the control of distinct enhancer elements. In addition, mef2
expression in the cardioblasts of the heart is dependent on at least two distinct enhancers, which are
active at different periods during embryogenesis. Mef2 expressing cells are coincident with those expressing Tinman. Notably, both Mef2 and Tinman expression are in four of six cardioblasts that are present per hemisegment. The complete overlap between the two expression patterns suggests that the activity of this enhancer element could be dependent on tinman function or under similar regulatory controls as is tinman. The cardiac enhancer that functions at later stages also drives mef2 expression in the caudal visceral mesoderm as well as in the somatic mesoderm. Moreover, multiple regulatory elements are
differentially activated for specific expression in presumptive muscle founders, prefusion myoblasts,
and differentiated muscle fibers. Taken together, the presented data suggest that specific expression of
the mef2 gene in myogenic lineages in the Drosophila embryo is the result of multiple genetic inputs
that act in an additive manner on distinct enhancers in the 5' flanking region (Nguyen, 1999).
The MADS-box transcription factor MEF2 is expressed specifically in developing cardiac, somatic, and visceral muscle cell lineages during Drosophila
embryogenesis and is required for myoblast differentiation and muscle morphogenesis. To define the mechanisms that regulate Mef2 transcription,
the Mef2 upstream region was analyzed for sequences sufficient to recapitulate the expression pattern of the gene in Drosophila embryos. Described here is a complex enhancer
located 5.8 kb upstream of the Drosophila Mef2 gene that controls transcription in cardial cells of the dorsal vessel, a subset of somatic muscle founder cells, and the
visceral muscle cells. The 237-bp cardial enhancer is located between -5907 to -5670 upstream of the Mef2 gene. The core of this enhancer contains two evolutionarily conserved binding sites for the homeodomain protein Tinman (Tin), expressed in
developing cardiac, somatic, and visceral muscle lineages. Both Tin binding sites are required for enhancer activity in all three muscle cell lineages. Whereas the
285-bp enhancer core alone is sufficient for expression in cardiac cells, expression in somatic founder cells and visceral muscle is dependent on the core enhancer
plus unique flanking sequences that include an evolutionarily conserved E box. These results reveal an essential role for Tin in the activation of Mef2 transcription in
multiple myogenic lineages and demonstrate that transcriptional activity of Tin is dependent on combinatorial interactions with other factors unique to different muscle
cell types (Cripps, 1999).
Expression of ladybird genes in the subset of cardioblast and pericardial cell
precursors is critically dependent on mesodermal tinman function, epidermal Wingless signaling and
the coordinate action of neurogenic genes. lb-expressing heart progenitors contribute to the increased number of cardiac precursor cells in Notch, Delta, Enhancer of split, mastermind, big brain and neuralized mutants. Negative regulation by hedgehog is required to restrict
ladybird expression to two out of six cardioblasts in each hemisegment. Overexpression of ladybird causes a hyperplasia of heart precursors and alters the identity of even-skipped-positive pericardial cells. Surprisingly, the number of eve-expressing pericardial cells is strongly reduced in overexpressors. These lb expressing cells are transformed into l-paracardial cells. Loss of ladybird function leads to the opposite transformation, suggesting that ladybird participates in the determination of heart lineages and is required to specify the identities of
subpopulations of heart cells. Both early Wingless signaling and ladybird-dependent late
Wingless signaling are required for proper heart formation. Thus, it is proposed that ladybird plays a dual role in cardiogenesis: (1) during the early phase, it is involved in specification of a segmental subset of
heart precursors as a component of the cardiogenic tinman-cascade and (2) during the late phase, it is needed for maintaining wingless activity and thereby sustaining the heart pattern process. These events result in a diversification of heart cell identities within each segment. Since tinman, bagpipe, S59 and ladybird genes are all part of the same homeobox gene cluster, it is likely that their association has to do with the orchestrated diversification of mesoderm (Jagla, 1997).
In an effort to isolate genes required for heart development and to further the understanding of cardiac specification at the molecular
level, PlacZ enhancer trap lines were screened for expression in the Drosophila heart. One of the lines generated in this screen, designated
B2-2-15, is particularly interesting because of its early pattern of expression in cardiac precursor cells, an expression pattern dependent on the
homeobox gene tinman, a key determinant of heart development in Drosophila. A gene was isolated and characterized in the vicinity of
B2-2-15 that exhibits an identical expression pattern to that of the reporter gene of the enhancer trap. apontic mutant embryos show distinct
abnormalities in heart morphology as early as mid-embryonic stages when the heat tube assembles: segments of heart cells (those
of myocardial and pericardial identity) are often missing. These abnormalities become obvious shortly before the assembly of the heart precursor cells at the dorsal midline. The defects in heart tube formation are seen with three markers: (1) Evenskipped, which is present in a subset of pericardial cells (EPC); (2) Mef2, which marks the cardial cells of the heart, and (3) Zfh-1, which is primarily present in the non-EPC pericardial cells. No obvious defects are observed in somatic and visceral muscle patterning, suggesting a specific requirement for apt in heart formation, as opposed to other mesodermal derivatives. Since the initial cardiac mesoderm seems to form normally in these mutants, it seems likely that apt is primarily required for a late differentiation step, such as the correct assembly of the heart tube. This would be consistent with a cell autonomous function of apt in the developing heart (Su, 1999).
During Drosophila embryogenesis, the beta3 tubulin gene is expressed in the visceral and somatic mesoderm as well as in the dorsal vessel. Transcription of the gene
is limited to four pairs of cardioblasts per segment. Its expression in the dorsal vessel (dv) is mediated by a 333-bp enhancer located upstream of
the gene (between -21705 and -21385 bp). The homeodomain protein Tinman is expressed in these cardioblasts, implying that Tinman might be a key regulator of the beta3 tubulin gene. Gel
retardation and footprint assays indeed has revealed two Tinman binding sites within the dv-specific enhancer. The relevance of the Tinman binding sites was analyzed in a
transgenic fly assay and distinct functions for both sites were observed. The BS(Tin-1460) site is absolutely required for expression in cardioblasts, while BS(Tin-1425) is
needed for high-level expression. Thus, these two Tinman binding sites act in concert to drive beta3 tubulin gene expression during heart development. Tinman
initially functions in the specification of visceral mesoderm and heart progenitors, but remains expressed in cardioblasts until dorsal closure. Overall, these data
demonstrate a late function for Tinman in the regulation of beta3 tubulin gene expression in the forming heart of Drosophila (Kremser, 1999a).
Genetic analyses indicate that tinman and D-mef2
act at early and late steps, respectively, in the cardiac lineage. D-mef2 expression in the developing heart requires a novel upstream enhancer containing two Tinman binding sites, both of which are essential for enhancer function in cardiac muscle cells. The upstream enhancer is located 5.4 kb upstream of the structural gene for D-mef2. Transcriptional activity of this cardiac enhancer is dependent on tinman function, and ectopic Tinman expression activates the enhancer outside of the cardiac lineage. These results define the only known in vivo target for transcriptional activation by Tinman and demonstrate that D-mef2 lies directly downstream of tinman in the genetic cascade controlling heart formation in Drosophila. Higher up in the cascade, both DPP and Wingless expression in the ectoderm are required for tinman expression in the dorsal mesoderm (Gajewski, 1997).
The Drosophila mef2 gene encodes a MADS domain transcription factor required for the
differentiation of cardiac, somatic, and visceral muscles during embryogenesis and the patterning of
adult indirect flight muscles assembled during metamorphosis. A prerequisite for Mef-2 function in
myogenesis is its precise expression in multiple cell types. Novel enhancers for
Mef-2 transcription in cardiac and adult muscle precursor cells have been identified and their
regulation by the Tinman and Twist myogenic factors have been demonstrated. However, these results
suggest the existence of additional regulators and provide limited information on the specification of
progenitor cells for different muscle lineages. The heart enhancer has been further characterized and shown to be
part of a complex regulatory region controlling the activation and repression of Mef-2
transcription in several cell types. The presence of two Tinman binding sites is necessary but not sufficient for enhancer function; additional sequences are required for cardial cell expression. The mutation of a GATA sequence in the enhancer changes its
specificity from cardial to pericardial cells. Also, the addition of flanking sequences to the heart
enhancer results in the expression of Mef-2 in a new cell type: the founder cells for a subset of body wall
muscles. Since tinman function is required for Mef-2 expression in both the cardial and founder cells,
these results define a shared regulatory DNA that functions in distinct lineages due to the combinatorial
activity of Tinman and other factors that work through adjacent sequences. The forced mesodermal expression of Twist causes a repression of the enhancer element in founder cells while allowing normal function in cardial cells. The analysis of
Mef-2-lacZ fusion genes in mutant embryos reveals that the specification of the muscle precursor
cells involves the wingless gene. Wg is required both in the formation of specific founder cells and in the specification of the progenitors of the cardial cells. Ectodermal cells must have a ventral identity for the formation of founder cells. These results demonstrate that the cell fate status of ectodermal cells adjacent to the domain of ventral founder cell specification is crucial for the proper formation of these cells. Mesodermal cell fate also depends on the activation of a receptor tyrosine kinase signaling pathway. Ectopic expression of an activated form of Ras1 throughout the mesoderm results in a substantial overproduction of the ventral founder cells as compared to control embryos. This signal may be transduced through the mesodermally-active EGF or FGF receptor tyrosine kinases (Gajewski, 1998).
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).
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).
During Drosophila embryogenesis, the homeobox gene tinman is expressed in the dorsal mesoderm where it functions in
the specification of precursor cells of the heart, visceral, and dorsal body wall muscles. The GATA factor gene pannier is
similarly expressed in the dorsal-most part of the mesoderm where it is required for the formation of the cardial cell lineage. Despite these overlapping expression and functional properties, potential genetic and molecular interactions between the two genes remain largely unexplored. pannier has been shown to be a direct transcriptional target of Tinman in the heart-forming region. The resulting coexpression of the two factors allows them to function combinatorially in the regulation of cardiac gene expression, and a physical interaction of the proteins has been demonstrated in cultured cells. Functional domains of Tinman and Pannier have been described that are required for their synergistic activation of the D-mef2 differentiation gene in vivo. Together, these results provide important insights into the genetic mechanisms controlling heart formation in the Drosophila model system (Gajewski, 2001).
Around the time of heart precursor cell specification, pnr
RNA is detected in the dorsal mesoderm in cells that also
express the tin gene. A pnr enhancer has been identified that is active in this heart-forming region and maps to a 457-bp DNA immediately
upstream of the gene. Because the enhancer contains two putative Tin recognition sites, the binding of Tim to this DNA was investigated. A GST-Tin
fusion protein was used in a gel-shift assay to test its ability
to bind to the Tin1 sequence TCAAGTG, a known recognition
element of the homeodomain protein in mesodermal enhancers of the D-mef2, tin, and b3 tubulin genes. Tin can bind specifically to the Tin1 consensus but not to a mutant version of the sequence. DNase
I protection assays were also performed with the fusion
protein on the pnr DNA, and two separate footprints were
obtained that correspond to the Tin1 and Tin2 sequences. These in vitro experiments demonstrate that Tin can recognize and bind to two sites within the pnr dorsal mesoderm enhancer, suggesting the regulatory DNA might
be a direct target of Tin transcriptional activity (Gajewski, 2001).
The defined pnr enhancer functions in the dorsal-most
cells of the mesoderm and in cells of the amnioserosa. To determine whether its
activity is regulated by Tin around the time of heart cell
formation, the expression of a pnr enhancer-lacZ fusion gene was monitored in tin gain and loss of function embryos.
Initially, the Gal4/UAS binary system was used to express tin throughout the mesoderm and mesectoderm under the control of the twi-Gal4 driver.
An expanded function of the enhancer was observed within
the mesoderm, coupled with ectopic activity in midline
cells of the central nervous system (CNS) due to the forced
expression of Tin. Conversely, in tin null
embryos, a complete absence of beta-galactosidase expression
is observed, which demonstrates a requirement of Tin
function for enhancer activity (Gajewski, 2001).
The D-mef2 gene is a direct transcriptional target of Tin
and Pnr in cardioblasts. A defined heart enhancer for the
gene contains a pair of essential Tin binding sites and a
required GATA element located in close proximity to one
of the Tin recognition sequences. Coexpression of the two factors in CNS midline cells results in the ectopic activation of the D-mef2 enhancer
normally expressed only in cardial cells. This result is compatible with the nuclear colocalization and physical interaction of Tin and Pnr in
cultured cells and provides an embryological assay for
identifying regions of the proteins that are essential for
their functional synergism. Nine deleted or point mutant
versions of Tin were tested in the synergism assay.
Tin(N351Q) has a single amino acid change in the homeodomain
and is unable to bind DNA. Coexpression of this mutant with wild-type Pnr fails to activate the D-mef2 enhancer. While a competent
homeodomain must be present in Tin for synergism with
Pnr, this region by itself is not sufficient as it fails in the
coactivation assay. The TN domain is a highly conserved 12 amino acid region found in Tin and most other NK-2 class proteins. A
10-amino acid deletion was made within this domain to
generate the Tin(1-35, 46-416) mutant, but this altered
protein is still able to function combinatorially with Pnr. Thus, the TN domain is dispensable in the synergism assay (Gajewski, 2001).
A transcriptional activation domain has been mapped to
the N-terminal 114 amino acids of Tin by using a cell
transfection strategy. To determine whether this region is required for functional interaction with Pnr, the Tin(111-416) deletion was generated and
tested. This truncated protein remained competent to synergize
with Pnr in the activation of the D-mef2 enhancer,
showing that the Tin transactivation domain is not
required. However, larger N-terminal deletions
result in Tin proteins that are functionally inactive. Specifically, removal of an additional 41 amino acids in Tin(152-416) has identified residues 111 to 151 as essential for Tin synergism with Pnr. The Tin(1-109, 192-416)
variant that contains the transactivation domain and homeodomain,
but lacks internal sequences including the required
41-amino acid region, is likewise nonfunctional in
the D-mef2 enhancer coactivation assay. Therefore,
these studies identify two distinct regions of Tin
needed for its combinatorial function with Pnr, an internal
segment of 41 amino acids adjacent to the transactivation
domain and the conserved homeodomain (Gajewski, 2001).
The ectopic activation assay was used to determine
those regions of Pnr that are essential for its functional
synergism with Tin. Six deleted or point mutant forms were
tested for enhancer activation in CNS midline cells. Pnr(1-457) represents a C-terminal truncation of the GATA factor that maintains zinc fingers 1 and 2, but
deletes two putative amphipathic a helices. This
C-terminal region has been shown to contain a transcriptional
activation domain, and the inability of the truncated protein to synergize with Tin demonstrates an essential requirement of this Pnr sequence. Pnr(E168K) and Pnr(C190S) contain single amino acid changes in the N-terminal zinc finger that correspond to mutations found in dominant alleles pnr. These mutations may affect the formation of the first zinc finger and result in proteins that
heterodimerize poorly with the Ush antagonist. However, two different dominant mutant Pnr proteins are able to synergize with Tin and direct D-mef2 expression in the CNS. In contrast, the mutation of a conserved
cysteine residue in zinc finger 2 in Pnr(C247S) inactivates
the protein in the synergism assay. This amino acid
change is likely to influence the formation of the
C-terminal zinc finger and identifies this region as an
essential functional domain of Pnr in the coactivation of
D-mef2. It is important to note that, although Pnr(1-457)
and Pnr(C247S) fail to synergize with Tin, they are competent
to bind the homeodomain protein in the GST pull-down
assay. In combination, these results
substantiate that intrinsic functional properties of Pannier
are perturbed in the two mutant forms of the GATA factor (Gajewski, 2001).
An unexpected finding of this work is that, while
the C-terminal transactivation domain of Pnr is required in
the combinatorial assay, the N-terminal transactivation
domain of Tin is not. One could envision a mechanism
wherein the presence of the single domain provided by Pnr
is sufficient for the activation properties of the heterodimeric
complex. Additionally, it can not be ruled out
that a second transactivation domain exists in Tin that was
not revealed previously in cell transfection studies. Also of note is the
nonrequirement of a proposed cardiogenic domain of Tin
that maps to the N-terminus of the protein. Specifically, Tin(111-
416) is competent to work with Pnr in the cooperative
activation of the D-mef2 heart enhancer, despite the absence
of residues 1 through 42. Instead, an internal 41-amino acid region between the
Tin transactivation domain and homeodomain has emerged
as a vital sequence for functional interaction with Pnr. A repressor activity of
Tin has been ascribed to residues 111 through 188, and it is plausible that,
based on the biological assay being used, multiple functional
characteristics may be uncovered within this region (Gajewski, 2001).
In the context of Tin's synergistic interaction with Pnr in
regulating a defined cardiac enhancer, association of the two through this domain may prevent Pnr from interacting with other proteins such as Ush. At the same time, because Tin has the potential to act as a transcriptional repressor
that recruits Groucho via this domain, the interaction of Tin and Pnr through the essential 111 to 151 subregion may be beneficial to Tin in its role as a
transcriptional activator by eliminating its possible association with inhibitory cofactors. Preliminary results suggest the molecular interaction of Tin and Pnr may be due in part to the presence of this domain (Gajewski, 2001).
Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and
Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting
both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects
of three signal-activated transcription factors (dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and
Tinman) on even-skipped, a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman
determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).
Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial
signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess
information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and
founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).
Given that the eve MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).
To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).
The finding that the three Wg-dependent factors, dTCF, Twi, and Tin, that directly regulate eve could explain why activated Ras is incapable of bypassing Wg in the induction of Eve progenitors. Therefore attempts were made to rescue Eve expression in wg mutant embryos by ectopically expressing Twi and Tin together with activated Ras. However, Eve progenitors were not recovered by this manipulation, perhaps due to the direct requirement of dTCF for eve MHE activity. While activated Arm can supply the missing downstream Wg transcription factor in this rescue experiment, Arm alone is capable of fully rescuing not only the Eve progenitors but also all of the Wg-dependent factors that regulate the MHE, including Twi, Tin, and the RTK/Ras pathway components. Thus, the combined effects of the MHE transcription factors could not be further evaluated in the absence of Wg signaling. Nevertheless, the rescue and enhancer mutagenesis data strongly support the involvement of Wg as a mesodermal competence determinant both upstream of the Ras pathway and directly (via dTCF) as well as indirectly (via Twi and Tin) in the transcriptional response to inductive RTK signaling (Halfon, 2000).
Since mutation of any single transcription factor binding site in the MHE causes only a partial loss of enhancer activity, it was considered whether different sites might function together synergistically. To test this possibility, binding site mutations for two different activators were combined. Simultaneous mutation of the dTCF and Twi1 sites led to reporter gene expression in approximately 5-fold fewer cells than would be expected from the additive independent effects of each mutation. A similar, though slightly less robust, synergy was observed when the dTCF and Ets3 mutations were combined (Halfon, 2000).
An assessment was made of whether ectopic coexpression of individual transcription factors or upstream signals would lead to cooperative effects on endogenous Eve expression. As previously reported, ectopic Wg has no effect on Eve expression at late stage 11, activated Ras1 induces extra Eve progenitors, and ectopic Wg plus activated Ras1 cause a lateral expansion of the progenitor clusters. When Twi is expressed using a twi-Gal4 driver, a few Eve-positive cells develop at ectopic positions. The magnitude of this effect is increased by coexpression of Wg and Twi, and even more so by coexpression of Twi with activated Ras1. The latter effect strikingly resembles that of Wg plus activated Ras1. With the simultaneous ectopic expression of Wg, Twi, and activated Ras1, Eve progenitors form an almost continuous anteroposterior stripe confined to the dorsal mesoderm. These results demonstrate a synergistic induction of Eve progenitors by various combinations of Wg, Twi, and activated Ras1 that parallels the synergistic loss of MHE activity seen by mutating the dTCF, Twi, and Ets binding sites. Taken together, these loss- and gain-of-function findings suggest that dTCF, Twi, and Pnt cooperate at the MHE to synergistically regulate Eve transcription and, by extension, to induce the specification of Eve progenitor fates (Halfon, 2000).
It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).
The beta3 tubulin gene of Drosophila is expressed in the major mesodermal derivatives during their differentiation. The gene is subject to complex stage- and
tissue-specific transcriptional control by upstream as well as downstream regions. Analysis of the vm1 enhancer, which is responsible for tissue-specific expression in
the visceral mesoderm and is localized in an intron, reveals a complex modular arrangement of regulatory elements. In vitro and in vivo experiments uncovered two
binding sites [termed UBX1 and UBX2, for the product of the homeotic gene Ultrabithorax(Ubx)] that are required for high-level expression in pPS6 and PS7.
Further analysis of the vm1 enhancer has revealed that deletion of a specific element, termed element 7 (e7), abolishes transcription of the lacZ reporter gene in all
parasegments except pPS6/PS7. Gel-retardation and footprint analysis has identified a binding site for the homeodomain protein Tinman, which is essential for the
specification of the dorsal mesoderm, within e7. Simultaneous deletion of two further sequence blocks in the vml enhancer, named elements 3 (e3), and 6 (e6),
results in a reduction analogous to that caused by removal of e7. The e6 sequence contains conserved motifs also found in the visceral enhancer of the Ubx gene.
It is therefore concluded that these elements act in concert with the Tinman binding site to achieve high expression levels. Thus the vm1 enhancer of the beta3 tubulin gene contains a complex array of elements that are involved in transactivation by a combination of tissue- and position-specific factors, including Tinman and UBX (Kremser, 1999b).
The subdivision of the lateral mesoderm into a visceral (splanchnic) and a somatic layer is a crucial event during early mesoderm
development in both arthropod and vertebrate embryos. In Drosophila, this subdivision leads to the differential development of gut
musculature versus body wall musculature. biniou, the sole Drosophila representative of the FoxF subfamily of forkhead domain genes, has a key role in the development of the visceral mesoderm and the derived gut musculature. biniou expression is activated in the trunk visceral mesoderm primordia downstream of dpp, tinman, and bagpipe and is maintained in all types of developing gut muscles (Zaffran, 2001).
bagpipe-expressing domains are defined by the intersecting dorsal activities of dpp/tin, which act positively, and segmentally modulated activities of wg/slp, which have repressing effects. bin also
requires tin activity for normal expression in the trunk visceral mesoderm primordia. Whereas bap expression is virtually absent in these cells upon loss of tin activity, residual bin expression is observed
in small clusters of cells. To test the possibility that residual
expression of bin in tin mutant embryos is due to
direct inputs from Dpp, bin expression was examined in embryos
in which dpp expression was induced ectopically in the entire
mesoderm. Ectopic dpp in a wild-type background, which causes
tin expression to be expanded ventrally, results
in an analogous expansion of the bin domains. Notably, ventral expansion of the bin domains is also observed upon ectopic dpp expression in the absence of tin activity, although the domains are narrow. Thus, Dpp is able to induce bin in the absence of tin, although tin activity is required for normal expression levels. The residual expression of bin in tin mutant embryos is unstable and not maintained in later stages of development (Zaffran, 2001).
Similar to tin, bap activity is also required for
normal bin expression. This result is in
agreement with the temporal sequence of bap and bin
expression and with the observed expansion of
bin throughout most of the dorsal mesoderm upon ectopic
bap expression in the mesoderm. These data suggest
that bin is furthest downstream within a mesoderm-intrinsic cascade of gene activation: twist -> tin -> bap -> bin.
Moreover, bin itself is required for normal bin
expression. Although bin expression initiates normally in
stage 10 bin mutant embryos, it disappears at early stage 11 in the trunk visceral mesoderm primordia of bin mutants,
except for those in PS1 and 2. bin
expression in these two parasegments is also less sensitive to the loss
of tin and bap activity. Furthermore, the
expression of bin in foregut, hindgut, and caudal visceral
mesoderm does not depend on any of the genes examined in the present study (Zaffran, 2001).
Whereas the above data show that maintenance of bin expression
in most of the presumptive trunk visceral mesoderm requires positive
autoregulation, they do not establish whether this autoregulatory loop
is direct or indirect. Of note, maintenance of bap during stage 11 (but not its initiation during stage 10) also requires bin activity. Therefore, it is possible that, at least during stage 11, bin and bap maintain
each other's expression through a cross-regulatory feedback loop (Zaffran, 2001).
A screen was performed to identify genes that are transcriptionally regulated by the homeodomain protein Tinman. Tin, a member of the NK family of homeodomain proteins, is required for organogenesis of the embryonic heart and visceral mesoderm. The screening method relies on genetic selection in yeast for a protein-DNA interaction. A library was screened that represents 15% of the Drosophila genomic DNA and six DNA fragments were obtained that satisfied genetic criteria in yeast for Tin binding sites. Most of the genomic DNA fragments were isolated multiple times. Sequence analysis has confirmed the presence of core recognition sites for NK class homeodomains in all of the fragments. To show that these fragments function as Tin-responsive enhancers in vivo, it was asked if they could drive expression of a reporter gene in patterns consistent with Tin regulation (Weiss, 2001).
The screen is surprisingly specific for genes regulated by Tinman (or closely related genes), as demonstrated both by the reporter-construct results and the genes that are located adjacent to the Tinman binding sites. Four fragments identified in the screen were inserted upstream of a lacZ reporter. Three of the four reporter constructs, tested as transgenes, are active in patterns consistent with Tin regulation. One fragment lies adjacent to jelly belly (jeb), a gene expressed in ventral, early mesoderm. The Tin binding site that led to the identification of jeb contains two Tin/NK2 class homeodomain recognition sites oriented as an imperfect inverted repeat. This genomic fragment was mapped to interval 48E9 of polytene chromosome 2R by in situ hybridization and based on the Drosophila genome sequence. The Tin binding sites lie adjacent to a P element insertion within a large intron of the jeb gene (Weiss, 2001).
jeb expression in tin mutant embryos is scarcely different from wild-type, though it may be somewhat reduced. Tin activation of jeb transcription is likely to be redundant with other regulators of mesoderm development. To test the sufficiency of Tin for activating jeb, embryos in which tin was ectopically expressed were assessed for ectopic jeb expression. Misexpression of tin in the ectoderm with an engrailed GAL4 driver does not alter jeb expression. Misexpression of tin throughout the mesoderm is sufficient to activate jeb expression at a late time (stage 12) when it is not expressed in wild-type embryos, and in cells where jeb is not normally expressed. A cofactor in the mesoderm may be required for Tin-mediated activation of jeb transcription. The expression domains of tin and jeb imply that Tin's role in the regulation of jeb is restricted to the earliest stages of jeb expression, since at late stage 10, Tin is only in dorsal mesoderm and Jeb is in ventral mesoderm (Weiss, 2001).
The ability of Tin to activate jeb transcription ectopically in the mesoderm implies that Tin plays an early and redundant function in the regulation of jeb. Other regulators that may play roles in the regulation of jeb include the bHLH protein Twist and the Pax domain protein Pox Meso (Weiss, 2001).
The Drosophila melanogaster genes midline and H15 encode predicted T-box transcription factors homologous to vertebrate Tbx20 genes. All identified vertebrate Tbx20 genes are expressed in the embryonic heart and both midline and H15 are expressed in the cardioblasts of the dorsal vessel, the insect organ equivalent to the vertebrate heart. The midline mRNA is first detected in dorsal mesoderm at embryonic stage 12 in the two progenitors per hemisegment that will divide to give rise to all six cardioblasts. Expression of H15 mRNA in the dorsal mesoderm is detected first in four to six cells per hemisegment at stage 13. The expression of midline and H15 in the dorsal vessel is dependent on Wingless signaling and the transcription factors tinman and pannier. The selection of two midline-expressing cells from a pool of competent progenitors is dependent on Notch signaling. Embryos deleted for both midline and H15 have defects in the alignment of the cardioblasts and associated pericardial cells. Embryos null for midline have weaker and less penetrant phenotypes while embryos deficient for H15 have morphologically normal hearts, suggesting that the two genes are partially redundant in heart development. Despite the dorsal vessel defects, embryos mutant for both midline and H15 have normal numbers of cardioblasts, suggesting that cardiac cell fate specification is not disrupted. However, ectopic expression of midline in the dorsal mesoderm can lead to dramatic increases in the expression of cardiac markers, suggesting that midline and H15 participate in cardiac fate specification and may normally act redundantly with other cardiogenic factors. Conservation of Tbx20 expression and function in cardiac development lends further support for a common ancestral origin of the insect dorsal vessel and the vertebrate heart (Miskolczi-McCallum, 2005).
In order to determine where mid and H15 fit in the genetic hierarchy controlling heart development, their expression was examined in several mutant backgrounds. The initiation of mid expression in the dorsal mesoderm in early stage 12 occurs after the expression of tin and pnr, as well as after the period of Wg signaling in the dorsal mesoderm, suggesting that mid and H15 are regulated downstream of the factors that confer cardiac fate. Indeed, the dorsal vessel expression of mid and H15 is completely lost in both wgcx4 and tinec40 mutant embryos, which fail to specify dorsal mesoderm. Embryos mutant for pnr have greatly decreased numbers of cardioblasts. Accordingly, mid and H15 expression is variably lost in pnrvx6 null mutant embryos, with most embryos completely lacking mid expression in the dorsal mesoderm. Ectopic expression of pnr throughout the mesoderm using the GAL4/UAS system is able to induce ectopic expression of mid and H15. These results indicate that the initiation of mid and H15 in the dorsal mesoderm is downstream of factors required for the specification of cardiac fate (Miskolczi-McCallum, 2005).
A second study by Qian (2005) provides more detailed information on a cellular fate switch accompanying loss- and gain-of-function of this gene pair. Referring to midline and H15 by the alternative name neuromancer (nmr) the Qian study shows that gene function causes a switch in cell fates in the cardiogenic region, in that the progenitors expressing the homeobox gene even skipped (eve) are expanded, accompanied by a corresponding reduction of the progenitors expressing the homeobox gene ladybird (lbe). As a result, the number of differentiating myocardial cells is severely reduced whereas pericardial cell populations are expanded. Conversely, pan-mesodermal expression of nmr represses eve, while causing an expansion of cardiac lbe expression, as well as ectopic mesodermal expression of the homeobox gene tinman. In addition, nmr mutants with less severe penetrance exhibit cell alignment defects of the myocardium at the dorsal midline, suggesting nmr is also required for cell polarity acquisition of the heart tube. In exploring the regulation of nmr, it was found that the GATA factor Pannier is essential for cardiac expression, and acts synergistically with Tinman in promoting nmr expression. Moreover, reducing nmr function in the absence of pannier further aggravates the deficit in cardiac mesoderm specification. Taken together, the data suggest that nmr acts both in concert with and subsequent to pannier and tinman in cardiac specification and differentiation. It is proposed that nmr is another determinant of cardiogenesis, along with tinman and pannier (Qian, 2005).
Integration of positive Dpp signals, antagonistic Wg inputs and mesodermal competence factors and thir impact of Bagpipe expression during Drosophila visceral mesoderm induction
Tissue induction during embryonic development relies to a significant
degree on the integration of combinatorial regulatory inputs at the enhancer level of target genes. During mesodermal tissue induction in
Drosophila, various combinations of inductive signals and
mesoderm-intrinsic transcription factors cooperate to induce the progenitors of different types of muscle and heart precursors at precisely defined positions within the mesoderm layer. Dpp signals are required in cooperation with the mesoderm-specific NK homeodomain transcription factor Tinman (Tin) to induce all dorsal mesodermal tissue derivatives, which include dorsal somatic muscles (the dorsal vessel and visceral muscles of the midgut). Wingless (Wg)
signals modulate the responses to Dpp/Tin along anteroposterior positions by
cooperating with Dpp/Tin during dorsal vessel and somatic muscle induction
while antagonizing Dpp/Tin during visceral mesoderm induction. As a result,
dorsal muscle and cardiac progenitors form in a pattern that is reciprocal to
that of visceral muscle precursors along the anteroposterior axis. The present
study addresses how positive Dpp signals and antagonistic Wg inputs are
integrated at the enhancer level of bagpipe (bap), a NK
homeobox gene that serves as an early regulator of visceral mesoderm
development. An evolutionarily conserved bap enhancer element requires combinatorial binding sites for Tin and Dpp-activated Smad proteins for its activity. Adjacent binding sites for the FoxG transcription factors encoded by the Sloppy paired genes (slp1 and slp2), which are direct targets of the Wg signaling cascade, serve to block the synergistic activity of Tin and activated Smads during bap induction. In addition, binding sites for yet unknown repressors are essential to prevent the induction of the bap enhancer by Dpp in the dorsal ectoderm. These data illustrate how the same signal combinations can have opposite effects on different targets in the same cells during tissue induction (Lee, 2005).
To investigate whether the bap regulators identified genetically, including tin, dpp, slp (downstream of wg) and biniou (bin), can act directly on the early TVM regulatory element of bap, DNaseI protection experiments with recombinant Tin, Bap, Smad (Mad and Medea), Slp and Bin proteins were performed on the 180 bp bap3.2.1 DNA sequence from D. melanogaster. The DNA footprinting results demonstrate that both Tin and Bap proteins can bind to the predicted Tin-binding site, which includes a perfect match to the canonical Tin-binding motif TCAAGTG. In addition to the Tin-binding site, a site with a TAAG core motif can strongly bind Bap but not Tin (CTTA in opposite strand; note that the same core motif is found in binding sites of a Bap ortholog, Nkx3.2). With regard to Dpp signaling mediators, there are five Mad-protected regions, three of which are also protected by recombinant Medea (Mad/Medea-1 to -3). Site 1 includes an AGAC motif that was initially identified as a Smad binding motif in vertebrates whereas sites 3-5 contain GC-rich sequences with CGGC motifs that were first shown to bind Smad proteins in Drosophila. Site 2 may be a combination of the two types (TGAC motif and CG-rich sequences). No clear correlation of either type of site was observed with the binding of Mad versus Medea. Finally, recombinant Slp proteins protect a wide stretch that includes an inverted repeat of core binding motifs for forkhead transcription factors (TAAACA), but extends further downstream. Slp can bind to tandem repeats of CAAA sequences, which are present in three copies in the 3' region of the protected region. Gel mobility shift and competition assays with Slp using wild-type oligonucleotides and a version in which the TAAACA motifs were mutated indicate that Slp can bind to both the TAAACA and the CAAA motifs with roughly equal affinity. In addition, the FoxF family protein Bin binds to the TAAACA inverted repeat region, but less well to the CAAA repeat region when compared with Slp. Taken altogether, these binding data are consistent with the hypothesis that the known mesodermal regulators of bap, namely Tin and Bin (and possibly autoregulatory Bap), as well as the signaling inputs from Dpp and Wg (through Smads and Slp, respectively) are integrated via direct binding to the early TVM enhancer of bap (Lee, 2005).
The present study describes an example of an enhancer whose response to Dpp is suppressed by Wg signals. A comparison of the functional organization of these enhancers provides new insight into molecular strategies of nuclear signal integration to produce differential developmental responses. The data show that bap is a direct target of Dpp signals. Thus, an indirect pathway of bap being activated solely by tin, whose mRNA expression is known to depend on Dpp inputs during the time of bap activation, can be ruled out. Rather, tin acts simultaneously and synergistically with Dpp. In fact, recent data with tin alleles lacking the Dpp-responsive enhancer show that bap can be induced in the absence of Dpp-induced tin products, as long as the twist-activated tin products are present. The molecular basis for this observed synergism of tin and dpp relies on the combinatorial binding of Tin and Dpp-activated Smad proteins to the bap enhancer. Several possible molecular mechanisms could underlie the strict requirement for combinatorial binding of Tin and Smads. For example, the relatively low binding affinity and specificity of Smads might be enhanced by bound Tin, which can engage in protein interactions with Mad and Medea. The combined presence of Tin and Smads in close vicinity or in complexes may also be a prerequisite for the assembly of higher order complexes with transcriptional co-activators such as CBP/p300. In addition, Tin may counteract the function of yet unknown repressors of nuclear Dpp signaling activity so that they can only repress in the ectoderm (Lee, 2005).
Unlike Dpp, Wg signals act indirectly upon the early bap enhancer. Previous genetic and molecular data have shown that Wg induces the expression of the forkhead domain-encoding gene slp via crucial dTCF/Lef-1 binding sites in both mesoderm and ectoderm. slp, in turn, functions as a repressor of bap. The present data show that slp products exert this function by direct binding to the Dpp-responsive bap enhancer, which obviously results in a suppression of the synergistic activity of bound Tin and Smad complexes. Slp proteins contain eh1 motifs that can potentially bind the Groucho co-repressor and Slp has known repressor activities in other contexts. In addition, the vertebrate counterpart of Slp, FoxG (BF-1), is known to interact with Groucho and histone deacetylases (Yao, 2001). Thus, it is proposed that Slp overrides nuclear Dpp signaling activities by dominantly establishing an inactive state of the chromatin at the bap locus (Lee, 2005).
Why would induction of tin and bap in the mesoderm require Tin as a co-factor of Smads, whereas in the ectoderm, which lacks Tin, the induction of tin and bap needs to be actively repressed? In the case of the tin enhancer, the ectodermal repressor elements are overlapping with the Tin-binding sites. Based upon this situation, a model is proposed in which the repressor would be present in both germ layers, but in cells of the mesoderm it is competed away from binding to the enhancer by Tin. This model is compatible with data showing that ectopic expression of Tin in the ectoderm is able to activate the Dpp-responsive enhancer of tin, even in the presence of the putative repressor binding elements. However unlike full-length Tin, an N-terminally truncated version with an intact homeodomain is not able to allow induction of the tin enhancer in the ectoderm. Furthermore, the putative repressor binding sites in the bap enhancer are separate from the Tin site. Hence, Tin does not compete for binding but may rather block or override the repressor factor(s) functionally. Thus, the positive activity of Tin would dominate over the negative action of this repressor in the mesoderm. By contrast, the repressing activity of Slp dominates over the positive action of Tin. Through this intricate balance of positive and negative switches, Tin could ensure that bap is induced by Dpp only in the mesoderm, while bound Slp prevents Tin from promoting Dpp inputs towards bap in striped domains within this germ layer. However, it can still not be fully explained why the absence of both the functional Tin and ectodermal repressor sites allows enhancer induction in the ectoderm, while preventing it in the mesoderm. The additional positive and negative binding factors involved will need to be identified to gain a full understanding of the germ layer-specific induction of these Dpp-responsive enhancers (Lee, 2005).
The bap enhancer described in this study represents the third example of well-characterized Dpp-responsive enhancers from mesodermal control genes. The other two are from tin, which is induced in the entire dorsal mesoderm, and eve, which is active in a small number of somatic muscle founder cells and pericardial progenitors in the dorsal mesoderm. The activities of the bap and eve enhancers along the anteroposterior axis are reciprocal, which is due to the fact that the eve enhancer requires inputs from Wg, whereas bap enhancer activity is suppressed by Wg. A comparison of the molecular architecture of these three enhancers reveals that they all share a number of important features. Most notably, all three enhancers feature several Tin- and Smad-binding sites in close vicinity that are essential for the activation of the enhancer in the mesoderm. Each enhancer includes both types of known Smad-binding motifs, which have 'AGAC' and 'CG'-rich cores, respectively. Hence, the basic activation mechanisms of each of the three enhancers downstream of Dpp are likely to be closely related. In the enhancers of both tin and bap, binding sites for a nuclear repressor of Dpp signals are key for the germ layer specificity of the inductive response. Although it is not known whether the same repressive mechanism operates at the eve enhancer, it is noted that motifs related to the presumed repressor binding motifs are present and their function can now be tested in vivo. As in the case of bap, the tin enhancer includes also additional sites that are required for Dpp-inducible enhancer activity, which may bind essential Smad co-factors. However, based upon the divergent sequences of these sites (C1 site in the bap and 'CAATGT' motifs in the tin enhancer), they appear to bind different types of factors in each case (Lee, 2005).
On top of this basic arrangement that allows the enhancer to be active in the dorsal mesoderm, the enhancers from bap and eve, but not tin, include binding sites that make them respond to Wg inputs in an opposite fashion. In the case of bap, Wg-induced Slp binds and dominantly suppresses the activity of bound Smad effectors. For the eve enhancer it has been proposed that there is an analogous repressive activity; however, in this case, it is exerted by bound Wg signal effectors, i.e., dTCF/Lef-1, in the absence of Wg signals. In the domains with active Wg signaling, the repressive activity of dTCF/Lef-1 is neutralized by the Wg signaling cascade, which allows the Dpp effectors to be active at the eve enhancer (since it lacks Slp binding sites). Through these switches, the bap and eve enhancers become induced in reciprocal AP patterns. In addition, the eve enhancer includes binding sites for activators and repressors downstream of receptor tyrosine kinases and Notch, respectively, which serve to restrict eve activity to specific subsets of cells within the domains of overlapping Dpp and Wg activities. Clearly, many of the molecular details still need to be clarified. Nevertheless, the basic principles of how differential inputs from inductive signals and tissue-specific activities can be integrated at the enhancer level to achieve distinct patterns of target gene expression during early tissue induction in the Drosophila mesoderm are now beginning to be understood (Lee, 2005).
Cardiac induction in Drosophila relies on combinatorial Dpp and Wg signaling activities that are derived from the ectoderm. Although some of the actions of Dpp during this process have been clarified, the exact roles of Wg, particularly with respect to myocardial cell specification, have not been well defined. The present study identifies the Dorsocross T-box genes as key mediators of combined Dpp and Wg signals during this process. The Dorsocross genes are induced within the segmental areas of the dorsal mesoderm that receive intersecting Dpp and Wg inputs. Dorsocross activity is required for the formation of all myocardial and pericardial cell types, with the exception of the Eve-positive pericardial cells. In an early step, the Dorsocross genes act in parallel with tinman to activate the expression of pannier, a cardiogenic gene encoding a Gata factor. Loss- and gain-of-function studies, as well as the observed genetic interactions among Dorsocross, tinman and pannier, suggest that co-expression of these three genes in the cardiac mesoderm, which also involves cross-regulation, plays a major role in the specification of cardiac progenitors. After cardioblast specification, the Dorsocross genes are re-expressed in a segmental subset of cardioblasts, which in the heart region develop into inflow valves (ostia). The integration of this new information with previous findings has allowed drawing a more complete pathway of regulatory events during cardiac induction and differentiation in Drosophila (Reim, 2005b).
In vertebrate species, genetic studies with loss-of-function alleles have
implicated Tbx1, Tbx2, Tbx5 and Tbx20 in the control of heart morphogenesis and the regulation of cardiac differentiation markers. In the case of Tbx5, a small number of cardiac differentiation genes have been identified as direct downstream targets. However, owing to the complexity of the system, the respective positions of these genes within a regulatory network during early cardiogenesis are still poorly understood (Reim, 2005b).
Drosophila offers a simpler system to study regulatory networks in cardiogenesis. The Tbx20-related T-box genes mid and H15 have been shown to play a role in cardiac development downstream of the early function of the NK homeobox gene tin and the Gata gene pannier (pnr). Whereas the role of these genes in the morphogenesis of the cardiac tube is minor, they are involved in processes of cardiac patterning and differentiation during the second half of cardiogenesis, which includes the activation of tin expression in myocardial cells (Reim, 2005a). The present report characterizes the roles of the Tbx6-related Dorsocross T-box genes (which may actually have arisen from a common ancestor of the vertebrate Tbx4, Tbx5 and Tbx6 genes), in Drosophila cardiogenesis. The Doc genes have a fundamental early role; they are required for the specification of all cardiac progenitors that generate pure myocardial and pericardial lineages. They are not required for generating dorsal somatic muscle progenitors and lineages with mixed pericardial/somatic muscle, even though their early expression domains also include cells giving rise to these lineages (Reim, 2005b).
The new information on the regulation and function of Doc fills a major gap
in the understanding of early Drosophila cardiogenesis. Previous data have shown that the combinatorial activities of Wg and Dpp are required for the formation of both myocardial and pericardial cells. In addition, the homeobox gene even-skipped (eve) is a direct target of the combined Wg and Dpp signaling inputs in specific pericardial cell/dorsal somatic muscle progenitors. Current data identify the Doc genes as downstream mediators and potential direct targets of combined Wg and Dpp signals during the induction of myocardial and Eve-negative pericardial cell progenitors. The induction of Doc expression by Wg and Dpp occurs concurrently with the induction of tin by Dpp alone, at a time when the mesoderm still consists of a single layer of cells. As a result, tin and Doc are co-expressed in a segmental subset of dorsal mesodermal cells that include the presumptive cardiogenic mesoderm. Conversely, in the intervening subset of dorsal mesodermal cells (the presumptive visceral mesoderm precursors) tin is co-expressed with bagpipe (bap) and biniou (bin), which are both negatively regulated by Wg via the Wg target sloppy paired (slp). Ultimately, these shared responses to Dpp, differential responses to Wg and the specific genetic activities of Doc versus bap and bin lead to the reciprocal arrangement of cardiac versus visceral mesoderm precursors in the dorsal mesoderm (Reim, 2005b and references therein).
Although the Dpp signaling pathway (and likewise, the Wg pathway) is
activated in both ectodermal and mesodermal germ layers, tin and bap respond to it only in the mesoderm. The germ layer-specific response of these genes to Dpp relies on two probably interconnected mechanisms. The first of these involves the additional requirement for Tin protein as a mesodermal competence factor for Dpp signals, which is initially produced in the mesoderm downstream of twist. The second involves the specific repression of the responses of tin and bap to Dpp in the ectoderm by yet unidentified factors that bind to the Dpp-responsive enhancers of these two genes. By contrast, the Doc genes are induced by Dpp and Wg with the same spatial and temporal expression patterns in both germ layers. This implies that the (yet unknown) Dpp and Wg-responsive enhancer(s) of the Doc genes are not subject to the ectodermal repressor activities acting on the tin and bap enhancers, and fits with the observation that induction of Doc in the mesoderm does not require Tin as a mesodermal competence factor. However, because of the distinct roles of Doc in the ectoderm and mesoderm, this situation also implies that Doc must act in combination with germ layer-specific co-factors to exert its respective functions. These data suggest that, in the early mesoderm, Doc acts in combination with tin (Reim, 2005b).
A key gene requiring combinatorial Doc and Tin activities for its
activation in the cardiac mesoderm is the Gata factor-encoding gene pannier (pnr). pnr expression is activated in the cardiac mesoderm shortly after the induction of Doc and tin, at a time when Doc expression has narrowed to the mesodermal precursors giving rise to pure cardiac lineages. The mechanisms restricting Doc expression to the cardiac mesoderm are currently not known, but as a consequence, pnr expression is also limited to the cardiac mesoderm. It is conceivable that Doc receives continued inputs during this period from the ectoderm through Dpp, whose expression domain narrows towards the dorsal leading edge by then. Together with the observed feedback regulation of pnr on tin and Doc, this situation leads to a prolonged co-expression of Tin, Doc and Pnr in the cardiac mesoderm of stage 11 to stage 12 embryos. Based upon the onset of the expression of early markers such as mid and svp, this is precisely the period when cardiac progenitors become specified (Reim, 2005b).
It is anticipated that the activation of some downstream targets in presumptive
cardiac progenitors requires the combination of two, or perhaps all three, of these cardiogenic factors. Potential target genes include mid, svp and hand. However, none of these candidates is essential for generating cardiac progenitors, although mid and svp are known to be required for the normal diversification of cardioblasts within each segment (Reim, 2005b).
The observation that forced expression of Pnr in the absence of any Doc
partially rescues cardiogenesis could indicate that the early, combinatorial functions of tin and Doc are primarily mediated by pnr. Alternatively, or in addition, this observation and the fact that a few cardioblasts can be generated without Doc could point to the existence of some degree of functional redundancy among these three factors. In the context of the latter possibility, it is tempting to speculate that the functional redundancy among T-box, Nkx and Gata factors during early cardiogenesis has further increased during the evolution of the vertebrate lineages. This would explain the less dramatic effects of the functional ablation of Tbx5, Nkx2-5 and Gata4/5/6 on vertebrate heart development as compared to the severe effects of Doc, tin or pnr mutations on dorsal vessel formation in Drosophila. Like the related Drosophila genes, these vertebrate genes are co-expressed in the cardiogenic region and developing heart of vertebrate embryos, which at least for Nkx2.5 and Gata6 also involves cross-regulatory interactions that reinforce their mutual expression (Reim, 2005b).
The observed co-expression of Doc, Tin and Pnr allows for the possibility
that, in addition to combinatorial binding to target enhancers, protein interactions among these factors play a role in providing synergistic activities during cardiac specification. Physical interactions of Tbx5 with Gata4 and Nkx2-5, as well between Nkx2-5 and Gata4 in vitro as well as synergistic activities cell culture assays have been demonstrated in mammalian systems and may be relevant to human heart disease. In Drosophila, the genetic interactions between Doc, tin and pnr observed both in loss- and gain-of-function experiments reveal similar synergistic activities of the encoded factors during early cardiogenesis. Altogether, these observations make it likely that these Drosophila factors also act through combinatorial DNA binding and mutual protein interactions to turn on target genes required for the specification of cardiac progenitors (Reim, 2005b).
Whereas pnr is expressed only transiently during early
cardiogenesis, tin and Doc continue to be expressed in developing myocardial cells, suggesting that they act both in specification and differentiation events. Recently it was shown that the T-box gene mid is required for re-activating tin in cardioblasts (Reim, 2005a). Of note, owing to the action of svp, Doc and tin are expressed in complementary subsets of cardioblasts within each segment. This mutually exclusive expression of tin and Doc implies that they are not acting combinatorially but, instead, act differentially during later stages of myocardial development. Hence, their activities could result in the differential activation of some differentiation genes such as Sulfonylurea receptor (Sur), which is specifically expressed in the four Tin-positive cardioblasts in each hemisegment (Nasonkin, 1999; Lo, 2001), and wingless (wg), which is only turned on in the two Doc-positive cells in each hemisegment of the heart that generate the ostia. Surprisingly, even the activation of some genes that are expressed uniformly in all cardioblasts has turned out to result from differential regulation within the Tin-positive versus Doc-positive cardioblasts. For example, regulatory sequences from the Mef2 gene for the two types of cardioblasts are separable and those active within the four Tin-positive cells are directly targeted by Tin. Likewise, regulatory sequences from a cardioblast-specific enhancer of Toll have been shown to receive differential inputs from Doc and Tin, respectively, in the two types of cardioblasts. In parallel with this differential regulation, it is anticipated that yet unknown differentiation genes are activated uniformly in all cardioblasts downstream of mid/H15 and hand. The integration of the new information on the roles of Doc in cardiogenesis has now provided a basic framework of signaling and gene interactions through all stages of embryonic heart development, which in the future can be further refined upon the identification of new components and additional molecular interactions (Reim, 2005b).
Early heart development in Drosophila and vertebrates involves the specification of cardiac precursor cells within paired progenitor fields, followed by their movement into a linear heart tube structure. The latter process requires coordinated cell interactions, migration, and differentiation as the primitive heart develops toward status as a functional organ. In the Drosophila embryo, cardioblasts emerge from bilateral dorsal mesoderm primordia, followed by alignment as rows of cells that meet at the midline and morph into a dorsal vessel. Genes that function in coordinating cardioblast organization, migration, and assembly are integral to heart development, and their encoded proteins need to be understood as to their roles in this vital morphogenetic process. The Toll transmembrane protein is expressed in a secondary phase of heart formation, at lateral cardioblast surfaces as they align, migrate to the midline, and form the linear tube. The Toll dorsal vessel enhancer has been characterized, with its activity controlled by Dorsocross and Tinman transcription factors. Consistent with the observed protein expression pattern, phenotype analyses demonstrate Toll function is essential for normal dorsal vessel formation. Such findings implicate Toll as a critical cell adhesion molecule in the alignment and migration of cardioblasts during dorsal vessel morphogenesis (Wang, 2005).
At the time dorsal-ventral polarity is established during early Drosophila development, Toll is associated with the plasma membrane around the entire syncytial blastoderm embryo. Thereafter, Toll exhibits zygotic expression on several cell surfaces, including a specific dorsal cell type in late-stage embryos. These were identified at first as leading-edge cells of the two-epidermal sheets moving toward the dorsal midline. Toll expression in dorsal aspects of the embryo has been reevaluated and, to the contrary, it has now been concluded the gene is expressed in cardioblasts of the developing and formed dorsal vessel (Wang, 2005).
Initially, Toll mRNA accumulation was analyzed by in situ hybridization, with gene transcripts first detected in dorsal cell populations in stage 12 embryos and later in two converging rows of cells during the process of dorsal closure. The likelihood of the Toll-positive cells being cardioblasts was strongly implied by the pattern of mRNA accumulation in stage 16 embryos. Toll expression was detected in roughly 50 cell pairs, and the organization of said cells was reminiscent of cardioblasts within structurally identifiable aorta and heart regions of the assembled dorsal vessel. The pattern of Toll protein expression was also investigated, with results comparable to those obtained in the RNA analysis. The transmembrane protein was detected in dorsal cells in late stage 12/early stage 13 embryos. Thereafter, it showed a clear presence on lateral surfaces of all cells aligned within two contiguous rows as they migrate toward the dorsal midline. By stage 16, the Toll-positive cells populate the core of the dorsal vessel, again within defined aorta and heart subregions. Toll was found exclusively on cardioblast surfaces, while organ-associated pericardial, lymph gland, and ring gland cells failed to express the protein. High-resolution analysis by confocal microscopy demonstrated Toll presence at lateral points of contact between all cardioblasts of the mature dorsal vessel (Wang, 2005).
Toll zygotic transcription is complex based on the numerous cell and tissue types that express the gene. Through efforts to identify a regulatory sequence controlling Toll expression in central nervous system (CNS) midline glial cells, Wharton (1993) located three regions upstream of the gene that possessed transcriptional enhancer activity. Relevant to the demonstration of Toll expression in the dorsal vessel, a 6.5-kb DNA was fortuitously found to direct lacZ reporter expression in all cardioblasts, and in pharyngeal and body wall muscles as well. Due to the interest in understanding how this expression might be regulated, the Toll cardioblast enhancer was delimited within the defined upstream region.
At first, the analysis involved testing Toll 5'-flanking DNAs for the ability to drive lacZ expression in embryos of transgenic strains. A 7.1-kb region located between ~9.3 and ~2.2 upstream of the gene showed strong enhancer function in all cardioblasts of the dorsal vessel. The DNA was subdivided into five overlapping segments, and only the most distal 1.7-kb DNA maintained cardioblast activity. Subsequently, five fragments spanning this 1.7-kb interval were tested for enhancer function, and dorsal vessel activity was mapped to a 305-bp sequence located between ~8.3 and ~8.0 relative to the Toll transcription start site. Consistent with the timing of Toll mRNA and protein accumulation in cardioblasts, the 305-bp enhancer becomes active during stage 12 and maintains its activity through all subsequent events of dorsal vessel morphogenesis. It is noteworthy that this small DNA also functions in amnioserosa cells from stage 11 through stage 15 (Wang, 2005).
Since Toll encodes a transmembrane protein with leucine-rich repeats in its extracellular domain, a prediction was made that Toll could function as a homophilic cell adhesion molecule, in addition to its well-characterized role as a signal-transducing receptor. In support of this hypothesis, induced expression of the protein in the nonadhesive Schneider 2 cell line causes cellular aggregation, with Toll accumulating at sites of cell-cell interaction. Such a localization property is characteristic of cellular adhesion molecules. Given the highly specialized localization, and structural and functional features of the protein, it is likely that Toll contributes prominently to the molecular environment that aligns and stabilizes cardioblasts on their path toward assembly within the dorsal vessel (Wang, 2005).
The observation of structurally defective dorsal vessels within Toll mutant embryos is consistent with the pattern of Toll expression in cardioblasts. D-MEF2 serves as a marker for all cardioblasts, from their early appearance through their organization within the mature organ. Based on D-MEF2 staining, it appears appropriate numbers of cardioblasts are specified in mutant embryos, but deviations are observed from the normal process of cardioblast alignment and synchronous migration as two contiguous rows of 52 cells. Several other markers for the formed dorsal vessel identified random gaps in the linear organ due to missing and/or abnormally located cardioblasts. Such cardiac phenotypes are reminiscent of those presented by faint sausage (fas) mutant embryos; mutations of the immunoglobulin-like cell adhesion molecule also led to cardioblast alignment problems. Whether Toll and Fas work in combination for the proper alignment and migration of these cells remains to be investigated. Additionally, while structural and phenotypic properties are consistent with its role as a cardioblast adhesion molecule, a function for Toll in mediating signaling events between neighboring cardiac cells cannot be ruled out. So far, no indicators exist for the latter possibility; it was not possible to demonstrate expression of potential Toll transcriptional effectors (Dorsal and Dif) in cells of the dorsal vessel. Either way, these molecular and genetic findings identify Toll as a vital player in dorsal vessel formation (Wang, 2005).
The regulation of Toll expression in cardioblasts was pursued due to an interest in further defining the transcriptional network controlling heart development in Drosophila. The studies demonstrated Toll heart expression is controlled by a 305-bp DNA located 8.0 kb upstream of the transcription start site. This regulatory module contains multiple binding sites for Doc T-box proteins and a single recognition site for the Tin homeodomain protein. The Toll dorsal vessel enhancer contains a single TCAAGTG sequence at nucleotides 163 to 169. The evidence is strong for the transcriptional enhancer being regulated by both of these cardiogenic factors. Doc and Tin are expressed in adjacent but nonoverlapping sets of cardioblasts within segments of the dorsal vessel; together, they make up the complete population of inner cardiac cells. A deletion of the distal part of the Toll 305-bp enhancer that removes the strong Doc-A footprint sequence, which likely binds multiple Doc molecules through T-box domain recognition of GTG motifs, eliminates enhancer function in Svp/Doc cells while maintaining activity in Tin cells. Systematically adding back T-box core binding elements to partially, then fully, reestablish the Doc-A binding site restores enhancer function in the Svp/Doc population (Wang, 2005).
As for Tin, mutation of its recognition element in the Toll 305 DNA leads to decreased and variable enhancer activity in both Tin and Svp/Doc cardioblasts. This result suggests that Tin is required not only for the activation of Toll expression in the four cardioblasts per hemisegment that are Tin positive after stage 12 but also for its initiation in all six cardioblasts in each hemisegment during early stage 12. The residual activity of the mutated Toll 305 DNA may reflect some degree of Tin regulation through cryptic, low-affinity binding sites present in the enhancer. Indeed, perusal of the Toll sequence identifies three candidate Tin elements that match the binding consensus at six of seven nucleotide pairs, and other Tin-regulated enhancers of genes such as D-mef2, ß3-tubulin, and pnr also employ more than one Tin binding site (Wang, 2005).
In contrast to the Toll 305 enhancer element, mutation of the exact Tin site in the Toll 258 DNA completely silenced the enhancer in the normally Tin-active cells. This result strongly implies that Tin, and at least one other factor working through the distal 47 bp of DNA, are required for activating the Toll gene. Candidates for such factors are the Doc T-box proteins, which are initially expressed in all cardioblast progenitors during mid stage 12, as well as products of the T-box genes H15 and Midline (Mid), which are expressed in all cardioblasts from mid stage 12 onward. Mid can bind to the same regions of Toll DNA as Doc, although the relevance of such interactions remains to be investigated. A combinatorial requirement for T-box proteins and Tin during the initiation and/or maintenance of Toll expression is further supported by the observation that derivatives of the enhancer containing only the Doc-A sequences fail to show activity in Svp/Doc cells. Together, these molecular data point to a mechanism wherein T-box proteins, in combination with Tin, initially activate the Toll gene in all cardioblast progenitors. After stage 12, Doc and Tin (perhaps in cooperation with H15 and/or Mid) activate Toll in two complementary subsets of cardioblasts of the dorsal vessel (Wang, 2005).
Unfortunately, a genetic requirement for these two factors in the regulation of the Toll enhancer cannot be proven at this time since Doc and tin mutant embryos fail to produce cardioblasts. Such an analysis could be attempted with the generation of specialized Doc or tin genetic backgrounds that allow for cardioblast specification early on, while lacking protein functions in later stages of dorsal vessel formation. However, forced-expression studies have demonstrated that individual expression of Tin or Doc2 leads to expanded enhancer activity, while simultaneous expression of the cardiac factors results in a robust activation of Toll transcription. These findings convincingly support the model of Doc and Tin being positive transcriptional regulators of the Toll dorsal vessel enhancer (Wang, 2005).
In addition to the demonstration of Doc and Tin as activators of Toll expression in the dorsal vessel, the regulatory analysis has generated important reagents that should facilitate the discovery of novel cardiac-functioning genes of Drosophila. That is, the Toll-cGFP and Toll-nGFP transgenes serve as sensitive markers for assessing distinct aspects of dorsal vessel morphogenesis in living animals. In stage 16 to 17 embryos and thereafter, Toll-cGFP expression can be used to monitor the formation and function of the three pairs of valvelike ostia within the heart region of the dorsal vessel. Likewise, Toll-nGFP can be used to determine the exact number and diversification status of cardioblasts, as larger nuclei are present within Tin-determined cells while smaller nuclei are found in Svp/Doc-determined cells. Such sensitive and easy-to-use reagents will be valuable in genomewide screens to discover new genes involved in Drosophila heart development (Wang, 2005).
The Hand gene family encodes highly conserved basic helix-loop-helix (bHLH)
transcription factors that play crucial roles in cardiac and vascular
development in vertebrates. In Drosophila, a single Hand
gene is expressed in the three major cell types that comprise the circulatory
system: cardioblasts, pericardial nephrocytes and lymph gland hematopoietic
progenitors. Drosophila Hand functions as a potent transcriptional activator, and converting it into a repressor blocks heart and lymph gland formation. Disruption of Hand function by homologous recombination also results in profound cardiac defects that include hypoplastic myocardium and a
deficiency of pericardial and lymph gland hematopoietic cells, accompanied by
cardiac apoptosis. Targeted expression of Hand in the heart
completely rescues the lethality of Hand mutants, and cardiac
expression of a human HAND gene, or the caspase inhibitor P35, partially rescues the cardiac and lymph gland phenotypes. These findings
demonstrate evolutionarily conserved functions of HAND transcription factors
in Drosophila and mammalian cardiogenesis, and reveal a previously
unrecognized requirement of Hand genes in hematopoiesis (Han, 2006).
The existence of hemangioblasts, which serve as common progenitors for hematopoietic cells and cardioblasts, has suggested a molecular link between cardiogenesis and hematopoiesis in Drosophila. However, the molecular mediators that might link hematopoiesis and cardiogenesis remain unknown. This study shows that the highly conserved bHLH transcription factor Hand is expressed in cardioblasts, pericardial nephrocytes and hematopoietic progenitors. The homeodomain protein Tinman and the GATA factors Pannier and Serpent directly activate Hand in these cell types through a minimal enhancer, which is necessary and sufficient to drive Hand expression in these different cell types. Hand is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes, and by Serpent in hematopoietic progenitors in the lymph gland. These findings place Hand at a nexus of the transcriptional networks that govern cardiogenesis and hematopoiesis, and indicate that the transcriptional pathways involved in development of the cardiovascular, excretory and hematopoietic systems may be more closely related than previously appreciated (Han, 2005).
To search for cis-regulatory elements capable of conferring the specific
expression pattern of Hand in cardioblasts, pericardial nephrocytes
and lymph gland hematopoietic progenitors, a series of reporter
genes were generated containing lacZ and the hsp70 basal promoter linked to
genomic fragments within a 13 kb genomic region encompassing the gene, and
reporter gene expression was examined in transgenic embryos. A 513 bp
minimal enhancer was identified referred to as Hand cardiac and hematopoietic (HCH)
enhancer, between exons 3 and 4 of the Hand gene. HCH is both necessary and sufficient to direct lacZ expression in
the entire embryonic heart and lymph gland in a pattern identical to that of
the endogenous Hand gene. Further deletions of this enhancer caused either a
partial or complete loss of activity. The 513 bp HCH enhancer
showed the same expression pattern in the heart and lymph gland as larger
genomic fragments that were positive for enhancer activity.
It is concluded that this enhancer fully recapitulates the temporal and spatial
expression pattern of Hand transcription in the distinct cell types
derived from the cardiogenic region (Han, 2005).
The homeobox protein Tinman is essential for the formation of
the cardiac mesoderm, from which the heart and blood progenitors arise. However, its
potential late functions remain unknown. It is believed that Tinman is not
required for the entirety of heart development in flies, because it is not
maintained in all the cardiac cells at late stages. The data reveal at least
one function for the late-embryonic Tinman expression, which is to maintain
Hand expression. The fact that ectopic Tinman can turn on
Hand expression dramatically in the somatic muscles is striking and
suggests the existence of a Tinman-co-factor in muscle cells that can
cooperate with Tinman to activate Hand expression; this co-factor
would not be expected to be expressed in pericardial cells or the lymph gland.
This co-factor should also be expressed in Drosophila S2 cells, since
transfected Tinman can increase activity of the HCH enhancer in S2 cells by
more than 100-fold. The generally reduced activity of the HCH enhancer that
results from mutation of the Tinman-binding sites also suggests that Tinman
activity is required to fully activate the Hand enhancer (Han, 2005).
Although Pannier and Serpent bind to the same consensus sites, these GATA
factors produce distinct phenotypes when overexpressed in the mesoderm.
Ectopic Pannier induces cardiogenesis, shown by the extra number of
cardioblasts and pericardial nephrocytes, but does not affect the lymph gland
hematopoietic progenitors. Ectopic Serpent, however, induces ectopic lymph
gland hematopoietic progenitors, but reduces the number of cardioblasts and
pericardial cells. Interestingly, pericardial cells with ectopic Serpent
expression have a tendency to form cell clusters such as the lymph gland
progenitors, suggesting a partial cell fate transformation. These results
suggest that Pannier functions as a cardiogenic factor, whereas Serpent
functions as a hematopoietic factor. Although both can activate Hand
expression, Pannier and Serpent activate the HCH enhancer in different cell
types. This assumption is also supported by the specific expression pattern of
Serpent and Pannier in late embryos. Serpent is detected specifically in the lymph gland hematopoietic progenitors but
not in any cardiac cells. Pannier expression in the cardiogenic region of late
embryos is not clear because of the interference by the high level Pannier
expression from the overlaying ectoderm. However, the lymph gland was examined
in late stage embryos and no Pannier expression was detected in these cells. Together with the evidence from loss-of-function and
gain-of-function experiments with Serpent, it is concluded that the HCH-5G-GFP
transgene is not expressed in the lymph gland because Serpent could not bind
to the mutant enhancer in the lymph gland cells; whereas the lack of
HCH-5G-GFP expression in cardiac cells is due to the inability of Pannier to
bind the mutant enhancer in these cardiac cells (Han, 2005).
Since tin and pnr are not expressed in all the cardiac cells
of late stage embryos but the Hand-GFP transgene is expressed in these cells,
it is likely that additional factors control Hand expression in the
heart. One group of candidates is the T-box family. Since Doc1, Doc2 and
Doc3 genes (Drosophila orthologs to vertebrate Tbx5) are
expressed in the Svp-positive cardioblasts where tin is not expressed, but H15
and midline (Drosophila orthologs to vertebrate Tbx-11) are expressed
in most of the cardiac cells in late embryos, it is likely that the T-box genes activate Hand
expression in cells that do not express tin and pannier.
However, the enhancer lacking GATA and Tinman sites has no activity,
indicating that the additional factors that may activate Hand
expression in the heart and lymph gland also requires these crucial Tinman and
GATA sites, probably through protein interaction between Tinman and the GATA
factors (Han, 2005).
In mammals, the adult hematopoietic system originates from the yolk sac and
the intra-embryonic aorta-gonad-mesonephros (AGM) region. The AGM region is derived from the mesodermal germ layer of
the embryo in close association with the vasculature. Indeed, the idea of the
hemangioblast, a common mesodermal precursor cell for the hematopoietic and
endothelial lineages, was proposed nearly 100 years ago without clear in vivo
evidence. Recently, this idea was substantiated by the identification of a
single progenitor cell that can divide into a hematopoietic progenitor cell in
the lymph gland and a cardioblast cell in the dorsal vessel in
Drosophila (Mandal,
2004). In addition to providing the first evidence for the
existence of the hemangioblast, this finding also suggested a close
relationship between the Drosophila cardiac mesoderm, which gives
rise to cardioblasts, pericardial nephrocytes and pre-hemocytes, and the
mammalian cardiogenic and AGM region, which gives rise to the vasculature
(including cardiomyocytes), the excretory systems (including nephrocytes) as
well as adult hematopoietic stem cells. In fact,
in both Drosophila and mammals, the specification of the cardiogenic
and AGM region requires the input of Bmp, Wnt and Fgf signaling. In
addition to the conserved role of the NK and GATA factors, GATA co-factors
(U-shaped in Drosophila and Fog in mice) also play important roles in
cardiogenesis and hematopoiesis in both Drosophila and mammals.
Recent studies have shown that the Notch pathway is required for both
cardiogenic and hematopoietic progenitor specification in Drosophila. It is
likely that Notch also plays an important role in mammalian hematopoiesis (Han, 2005).
This study found that Drosophila Hand is expressed in
cardioblasts, pericardial nephrocytes and pre-hemocytes, and is directly
regulated by conserved transcription factors (NK and GATA factors) that
control both cardiogenesis and hematopoiesis. The bHLH transcription factor
Hand is highly conserved in both protein sequence and expression
pattern in almost all organisms that have a cardiovascular system. In mammals,
Hand1 is expressed at high levels in the lateral plate mesoderm, from
which the cardiogenic region and the AGM region arise, in E9.5 mouse embryos.
Functional studies of Hand1 and Hand2 using knockout mice
have demonstrated the essential role of Hand genes during cardiogenesis,
whereas the functional analysis of Hand genes during vertebrate hematopoiesis
has not yet been explored. It will be interesting to determine whether
mammalian Hand genes are also regulated in the AGM region by GATA1, GATA2 and
GAT3 (vertebrate orthologs to Drosophila Serpent), and whether they
play a role in mammalian hematopoiesis (Han, 2005).
In summary, this study places Hand at a pivotal point to link the
transcriptional networks that govern cardiogenesis and hematopoiesis. Since the Hand gene family encodes highly conserved bHLH transcription factors expressed in
the cardiogenic region of widely divergent vertebrates and probably in the AGM
region in mouse, these findings open an avenue for further exploration of the
conserved transcriptional networks that govern both cardiogenesis and
hematopoiesis, by studying the regulation and functions of Hand genes in
vertebrate model systems (Han, 2005).
U-shaped is a zinc finger protein that functions predominantly as a negative transcriptional regulator of cell fate determination during Drosophila development. In the early stages of dorsal vessel formation, the protein acts to control cardioblast specification, working as a negative attenuator of the cardiogenic GATA factor Pannier. Pannier and the homeodomain protein Tinman normally work together to specify heart cells and activate cardioblast gene expression. One target of this positive regulation is a heart enhancer of the Drosophila mef2 gene and U-shaped has been shown to antagonize enhancer activation by Pannier and Tinman. Protein domains of U-shaped required for its repression of cardioblast gene expression were mapped. Such studies showed GATA factor interacting zinc fingers of U-shaped are required for enhancer repression, as well as three small motifs that are likely needed for co-factor binding and/or protein modification. These analyses have also allowed for the definition of a 253 amino acid interval of U-shaped that is essential for its nuclear localization. Together, these findings provide molecular insights into the function of U-shaped as a negative regulator of heart development in Drosophila (Tokusumi, 2007).
Through the use of an established assay to monitor Pannier-dependent cardioblast gene activity, and the generation and analysis of 20 different versions of the U-shaped protein, six U-shaped domains required for its repression of mef2 gene expression were identified. Three previously identified GATA-interacting zinc fingers of U-shaped are critical for this inhibitory property, which likely reflects the necessity of multiple zinc fingers forming a strong and stable interaction with the Pannier GATA factor. Whether Pannier-U-shaped complex formation interferes with the physical interaction of Pannier and Tinman in the synergistic activation of D-mef2 target sequences remains to be determined (Tokusumi, 2007).
U-shaped may also directly antagonize Pannier function as has been shown in the process of sensory bristle formation. Heterodimerization of U-shaped with Pannier converts the GATA transcriptional activator into a transcriptional repressor, an event that leads to the non-activation of target genes such as ac, sc, and wg in the dorsal notum of the wing disc. It is noteworthy that the results demonstrated the requirement of a binding site for the CtBP transcriptional co-repressor protein. In the context of the cardiogenic mesoderm, the combination of Pannier, U-shaped, and CtBP may prevent mesodermal cells from initiating gene expression programs needed for the specification of the cardioblast fate. In contrast, the combination of Pannier, Dorsocross, and Tinman is known to activate a regulatory network programming heart cell specification and cardioblast differentiation. Additional studies will be needed to elucidate the potential role of CtBP as an antagonist of cardiac gene expression and heart development. If U-shaped-CtBP interaction plays a crucial inhibitory role, then one would predict comparable dorsal vessel phenotypes for CtBP and U-shaped in loss- and gain-of-function genetic backgrounds (Tokusumi, 2007).
Finally, these studies have defined a 253 amino acid region required for nuclear localization of U-shaped. Within this interval, two highly basic amino acid sequences have been defined as being essential for U-shaped ability to inhibit Pannier-mediated cardiac gene expression. Perhaps, these motifs are required to facilitate the binding and stable interaction of co-repressor proteins with U-shaped. Another possibility is that these sequences serve as sites for post-translational modification, such as acetylation and/or methylation. Selective protein modification(s) may be a requisite for U-shaped to act as a negative modulator of Pannier transcription factor function during cardiogenesis in Drosophila (Tokusumi, 2007).
The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. Focus was placed on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with KATP (ATP-sensitive potassium) channels, which are involved in metabolic homeostasis. Experimental evidence is provided that Tinman directly regulates dSUR expression in the developing heart. A cis-regulatory element was identified in the first intron of dSUR that contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly’s myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacing-induced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance (Akasaka, 2006; full test of article).
Because cardiac dSUR expression depends on Tin, 40 kb of the dSUR locus was scanned for Tin consensus binding sites (TNAAGTG). Three large genomic fragments were chosen based on the high density of potential Tin-binding sites (En1, 4,095 bp; En2, 2,151 bp; and En3, 2,291 bp). The enhancer activity of these En fragments was then examined in transgenic flies. Two fragments located upstream of the ATG start (En1 and En2) do not show any reporter activity in the embryonic heart. In contrast, En3 exhibits a pattern of reporter gene expression identical to the endogenous dSUR pattern. This En3 fragment is downstream of the ATG start and contains six Tin sites. To determine whether these Tin sites are required for cardiac expression, they were mutated. Of the mutated Tin sites, only a mutated T3 site reduced the enhancer’s transcriptional activity. Mutations in both T2 and T3 (241 bp apart) abolished reporter gene expression in the cardiac progenitor cells, suggesting that Tin is capable of directly activating dSUR expression in the appropriate myocardial cells. Shorter fragments (S, 890 bp; SS, 359 bp; and SSS, 297 bp) containing both T2- and T3-binding sites were tested for enhancer activity. These three fragments mimicked the cardiac dSUR expression and showed a similar expression level as seen with En3. Within the context of the short SSS fragment, the T3-binding site is absolutely essential for reporter gene activation. The En3 fragment was also scanned for Mad/Media (Dpp pathway)-binding sites (GCCGCGACG). No Mad/Media sites were found with appreciable conservation within this enhancer, which is consistent with Dpp signaling only indirectly regulating dSUR expression, possibly by means of tin. However, a direct regulation by Dpp by means of degenerate or not well conserved sites cannot be excluded (Akasaka, 2006).
An EMSA was performed to test whether Tin can directly bind to the T3 site. A DNA template (28 bp) composed of dSUR genomic sequence containing the T3-binding site produced a specific Tin-binding complex. Thus, Tin can directly associate with the T3 element in dSUR, which is consistent with the possibility that dSUR expression is directly controlled by Tin (Akasaka, 2006).
The Tin expression pattern varies by developmental stage, and, likewise, its downstream target genes may also change during development. In vertebrates, GATA-4 provides the binding efficiency to Nkx2.5 in cardiomyocytes; therefore, these two transcription factors can act cooperatively to activate cardiac genes. Similarly, the Drosophila counterparts Pnr and Tin physically interact and synergistically control cardiac gene expression of genes such as Dmef2. To further characterize the role of Pnr in dSUR activation, Pnr was expressed panmesodermally and the expression of dSUR was compared to that of dHand, which marks all cardiac lineages. Panmesodermally expressed Pnr activates both ectopic dHand and dSUR expression but only to a moderate extent. In contrast, a dominant-negative Pnr (Pnr-EnR) did not induce, and instead reduced, dSUR and dHand expression. Moreover, both dSUR and dHand were strongly activated when tin and pnr were coexpressed, suggesting that, like dHand, dSUR activation depends on genetic synergy between Tin and Pnr (Akasaka, 2006).
Next, whether a reduction of Pnr activity could be compensated for by overexpression of tin was examined. tin and pnr-EnR were co-expressed throughout the mesoderm and it was found that the reduced dSUR and dHand expression, which was due to Pnr-EnR, was not rescued by forced panmesodermal tin expression. This finding suggests that dSUR activation requires not only Tin but also Pnr activity (Akasaka, 2006).
Furthermore, Pnr consensus binding site (WGATAR) was sought within the En3 fragment to explain synergistic activation by Tin and Pnr. There are two well conserved Pnr sites in the SS fragment. However, when the enhancer activity was examined when both of these Pnr consensus sites were mutated [SS(P2P3)], it was found that this enhancer was equivalent to the WT SS fragment. This finding implies that Pnr could bind to Tin directly or to other nonconsensus Pnr-binding sites, such as TGATA (which exists in the SSS fragment), to activate dSUR expression in the embryonic heart (Akasaka, 2006).
To address the possibility that Tin and Pnr may be acting in a complex in regulating cardiac dSUR transcription, an in vitro reporter assay with a luciferase plasmid was used, in which expression was driven by six concurrent T3. Cotransfection of the T3 reporter plasmid with Tin but not the Pnr expression vector into Drosophila Schneider cells resulted in a 3-fold activation of luciferase activity compared with the reporter construct alone. In contrast, when Tin and Pnr were cotransfected, the luciferase activity was elevated 9-fold compared with the reporter construct alone (or with a mutant T3-binding site), suggesting that Pnr acts as a cofactor with Tin to synergistically activate dSUR transcription (Akasaka, 2006).
It has been shown that in corpora cardiaca (CC) cells of Drosophila, dSUR controls glucose homeostasis by increasing secretion of adipokinetic hormone (AKH) in response to low glucose concentration in the hemolymph. Evidence indicates that AKH release likely is increased by the SUR inhibitor sulfonylurea and is decreased by ectopic expression of constitutively active (and thus ATP-independent) ion channel Kir2.1, suggesting striking parallels between endocrine cells in Drosophila and mammals in controlling blood glucose. Therefore, the role of dSUR was examined in cardiac physiology and heart homeostasis in adults. The findings suggest that there are striking functional similarities between Drosophila and mammalian SUR in heart function. In the mammalian heart, there are two types of KATP channels, sarcKATP and mitochondrial KATP, which are candidate regulators of acute hypoxia and IPC. Impairing sarcKATP channel activity by genetic manipulation of mouse Kir6.2 results in compromised recovery of contractile function after hypoxia. The data are consistent, with dSUR in Drosophila providing a similar protective mechanism against hypoxia. Moreover, a recent study in goldfish KATP channel function revealed that the involvement of KATP in IPC is widely conserved, including in highly hypoxia-tolerant species (Akasaka, 2006).
To further address dSUR function, external electrical pacing of the heart was performed in dSUR knock-down mutants. Rapid electrical pacing per se is a nonhypoxic stimulus that may induce an IPC effect in mammals by activating KATP channels. Indeed, Kir6.2 mutant hearts exhibit diminished electrical tolerance against catecholamine-induced ventricular arrhythmia because of a failure to achieve action potential shortening and by causing early after-depolarization. Thus, the elevated heart failure rate in dSUR knock-down hearts may be due to KATP channel insufficiency. Interestingly, IPC is no longer observed in older human patients, and in female guinea pigs, SUR2A expression is reduced in old ventricular tissue compared with young ventricular tissue. Moreover, human SUR2 mutations found in two independent families were recently shown to cause dilated cardiomyopathy, with an onset around middle age. These mutations result in the structural abnormalities of the KATP channel and impair the ATP-dependent channel gating. Patients carrying these mutations showed ventricular tachycardia with normal coronary angiography, suggesting that human cardiomyocyte KATP channels play a role in maintaining membrane electrical stability and that the reduction of the KATP channel activity causes electrical disturbance, especially in older hearts. In this study it was observed that pacing-induced heart failure steeply increases in aging flies, which can be reversed by exposure to the KATP channel activator pinacidil. These observations, together with the drastically reduced dSUR expression in old flies, suggest that dSUR serves as an indicator of cardiac aging. Given the experimental advantages of Drosophila, such as a small genome size and short life span, dSUR and cardiac aging provide a unique model not only for assessing the control of physiological heart functions, such as the response to hypoxia, but also for the analysis of age-related human diseases (Akasaka, 2006).
A complex regulatory cascade is required for normal cardiac development, and many aspects of this network are conserved from Drosophila to mammals. In Drosophila, the seven-up (svp) gene, an ortholog of the vertebrate chick ovalbumin upstream promoter transcription factors (COUP-TFI and II), is initially activated in the cardiac mesoderm and is subsequently restricted to cells forming the cardiac inflow tracts. This study investigated svp regulation in the developing cardiac tube. Using bioinformatics, a 1007-bp enhancer of svp was identified which recapitulates its entire expression in the embryonic heart and other mesodermal derivatives; this enhancer is initially activated by the NK homeodomain factor Tinman (Tin) via two conserved Tin binding sites. Mutation of the Tin binding sites significantly reduces enhancer activity both during normal development and in response to ectopic Tin. This is the first identification of an enhancer for the complex svp gene, demonstrating the effectiveness of bioinformatics tools in assisting in unraveling transcriptional regulatory networks. These studies define a critical component of the svp regulatory cascade and place gene regulatory events in direct apposition to the formation of critical cardiac structures (Ryan, 2007).
In order to identify the svp cardiac enhancer (SCE), an initial goal was to identify a transcription factor whose function was required for svp expression in the dorsal vessel. Published data demonstrated that tinman (tin) and seven-up (svp) are co-expressed at stage 11, and become mutually exclusive shortly thereafter. Given the temporal and spatial coincidence of tin and svp expression during early cardiogenesis, and given the role that Tin plays in the expression of other important cardiac genes, it was hypothesized that tin function might be required for svp expression. Thus, Svp accumulation was evaluated in heterozygotes and homozygotes for the tin null allele tinEC40. svp expression was monitored in this experiment using the svp-lacZ enhancer trap line. The normal complement of seven bilateral pairs of Svp cells was visible in heterozygous embryos, whereas homozygous tinEC40 sibling embryos lacked cardiac svp expression, although Svp was still present in the ring gland. This result supported Tin as an upstream regulator of svp, however, whether Tin itself bound to the svp enhancer was to be determined. The possibility was considered that svp expression was not initiated in tin mutants simply because cardiac specification had failed to occur. However, since svp expression is initiated relatively early during cardiac specification, it was felt that Tin would still be a strong candidate activator (Ryan, 2007).
Given the dependence of svp expression on the presence of Tin, the svp gene was examined for consensus Tin binding sites. Seven sites, all located within the very large first intron of the svp-RC transcript variant, were identified. Since Tin targets have been shown to contain two closely apposed Tin binding sites, focus was placed upon the genomic regions where at least two sites lay within 300 bp of each other. Six of these putative Tin binding sites were present as three such pairs (Ryan, 2007).
It was then determined if any of the three genomic regions containing putative Tin binding sites were conserved in other Drosophila species. Only one region, encompassing the Tin sites at 8092711/8092853, showed strong sequence similarity between species. When compared with five additional Drosophila species, the putative Tin binding sites within this region were 100% conserved. To determine if Tin protein can bind to the putative Tin sites, an electrophoretic mobility shift assay, using Tin protein and radioactively labeled double-stranded oligonucleotides corresponding to each of the two putative Tin sites was performed. Tin protein, generated in vitro, bound to both of the radioactive DNA sequences, more strongly in the case of the Tin 1 site. Binding was effectively competed with identical respective unlabeled sequences, but not with respective mutated sequences. The higher affinity of the Tin1 binding was further reflected in the corresponding competition assay, in which a light band was still evident when competed with wild-type probe. These results further supported the notion that Tin might directly regulate svp gene expression via this genomic region (Ryan, 2007).
This study showd that Tin is an essential regulator of svp gene expression in the cardiac mesoderm, via activation of a an ~1 kb enhancer (termed the SCE) located in the first intron of the svp gene. Thus, it appears that Tin mediates this 'cardiac context' (Ryan, 2007).
The role of Tin in the initial activation of svp reflects the critical role tin plays in cardiac development in Drosophila. tin function is essential for cardiac specification, and a number of genes expressed in the dorsal vessel have been identified as direct transcriptional targets of Tin. It is anticipated that the SCE will ultimately provide greater insight into developmental patterning processes, since further analysis of the enhancer should identify how both Hox and Hh signals impact svp gene expression, as a model of how they impact cardiac patterning in general (Ryan, 2007).
Once svp expression is initiated, it soon becomes mutually exclusive with tin expression, and a svp-lacZ enhancer trap line is active in the Svp cells all through development to adulthood. In situ hybridization of SCE-lacZ embryos showed that the enhancer is active during embryogenesis through stage 14, although the activity had waned by stage 16. Thus, the SCE is responsible for the initial activation of cardiac svp gene expression at stages 12 to 14, yet other regulatory sequences mediate subsequent sustained svp expression. Since SCE activity is strong at stage 14, a time at which tin and svp expression do not overlap, it is reasonable to suggest that additional enhancer sequences must contribute to this period of expression. In contrast, mutation of the Tin sites also affects enhancer activity at stage 14, when Tin is absent from Svp cells. How can the integrity of Tin sites be required for enhancer activity at a stage when Tin is no longer present? One possibility is that initial binding of Tin to the enhancer might induce epigenetic changes to the genomic region, which can facilitate subsequent enhancer activity. In support of this notion, Tin has been shown to interact directly with the transcription cofactor P300 (Ryan, 2007).
Previous studies have demonstrated the importance of both the svp gene and its vertebrate ortholog, COUP-TFII, in cardiac development. Each factor is expressed in the cardiac inflow tracts: in the case of Drosophila, the inflow tracts are represented by the ostia, which form from the Svp cells and which require svp function for their formation; in vertebrates, COUP-TFII is expressed in and required for the formation of the atria. While neither upstream regulatory factors nor downstream targets of svp and COUP-TFII have been characterized to date, it is reasonable to speculate that such genes showing conserved functions might also share common upstream regulators. Thus, it is predicted that activation of COUP-TFII in vertebrate atrial cells might be mediated at least in part by NKX2.5, although no studies have directly assessed the expression of COUP-TFII in NKX2.5 mutants. Since this study used the dependence of svp expression upon tin function to predict the location of the SCE, such an approach might also be used to identify the COUP-TFII cardiac enhancer based upon the presence of conserved binding sites for NKX2.5. Given that COUP-TFII lies within a large, gene-poor genomic region in both mice and humans, this approach may facilitate the still significant task of illuminating the genetic regulation of COUP-TFII (Ryan, 2007).
Sandmann, T., et al. (2007). A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev. 21: 436-449. Medline abstract: 17322403
Embryogenesis is controlled by large gene-regulatory networks, which generate spatially and temporally refined patterns of gene expression. This study reports the characteristics of the regulatory network orchestrating early mesodermal development in the fruitfly, where the transcription factor Twist is both necessary and sufficient to drive development. Through the integration of chromatin immunoprecipitation followed by microarray analysis (ChIP-on-chip) experiments during discrete time periods with computational approaches, >2000 Twist-bound cis-regulatory modules (CRMs) were identified and almost 500 direct target genes. Unexpectedly, Twist regulates an almost complete cassette of genes required for cell proliferation in addition to genes essential for morophogenesis and cell migration. Twist targets almost 25% of all annotated Drosophila transcription factors, which may represent the entire set of regulators necessary for the early development of this system. By combining in vivo binding data from Twist, Mef2, Tinman, and Dorsal an initial transcriptional network was constructed of early mesoderm development. The network topology reveals extensive combinatorial binding, feed-forward regulation, and complex logical outputs as prevalent features. In addition to binary activation and repression, it is suggested that Twist binds to almost all mesodermal CRMs to provide the competence to integrate inputs from more specialized transcription factors (Sandmann, 2007).
ChIP-on-chip was performed at two consecutive developmental time periods: 2-4 h (stages 5-7) and 4-6 h (stages 8-9), covering the stages of gastrulation, mesoderm expansion, migration, and early subdivision into different primordia. For each time period, four independent ChIPs were performed using two different anti-Twist antibodies to reduce possible off-target effects (Sandmann, 2007).
To systematically identify Twist-bound regions in an unbiased, global manner, a high-density microarray tiling across the Drosophila melanogaster genome was designed with ~380,000 60mer oligonucleotide probes. Twist binds to E-box motifs: As a degenerate E-box (CANNTG) is expected to occur every ~256 base pairs (bp) in the Drosophila genome, a 60mer oligonucleotide was designed for each E-box motif within the nonrepetitive, noncoding regions of the genome. This design made no assumptions about the specificity of the E-box bound by Twist, yet ensured all putative E-boxes were covered and that each Twist-bound sequence was detected by at least two neighboring 60mers (Sandmann, 2007).
These experiments identified 2096 nonoverlapping genomic regions significantly bound by Twist within one or both developmental time periods. This set includes all known Twist-bound enhancers tested, e