Dorsocross1, Dorsocross2 and Dorsocross3
Northern analysis with gene-specific probes shows that all three Doc genes display similar expression profiles during development with maximal levels occurring between 2 and 12 hours of embryonic development and lower levels during late embryonic, larval and pupal stages. The only significant difference among the three genes in this assay is the expression of Doc1 mRNA in adult males: this was not observed for Doc2 and Doc3 (Reim, 2003).
The spatial expression patterns of Doc products in embryos were examined by whole-mount in situ hybridization with gene-specific probes and whole-mount immunocytochemistry using antibodies raised against the unique C-terminal regions of the Doc proteins. Since all three genes have essentially identical expression patterns (with some minor differences regarding the relative levels of expression in different tissues), they will be collectively referred as 'Doc genes'. As expected, Doc proteins are exclusively nuclear during interphase (Reim, 2003).
The initial expression of Doc genes is observed at the cellular blastoderm stage in a transverse stripe encompassing the dorsal ~40% of the embryonic circumference within the prospective head region. A short time later, a narrow longitudinal stripe of expression appears, which ultimately extends all along the dorsal midline of the embryo, and the joint domains form a cross-shaped pattern of Doc expression in dorsal areas of the early embryo. The domain of the transverse stripe is located anterior to the cephalic furrow, formed during gastrulation and largely corresponds to procephalic neuroectoderm. The cells of this domain continue Doc expression until stage 11, when the segregation of procephalic neuroblasts is completed. By contrast, the cells from the dorsal longitudinal domain within the trunk region give rise to amnioserosa, which maintains strong Doc expression until stage 15. In addition, the cells from the anterior and posterior termini within this longitudinal stripe contribute to regions of the anterior and posterior digestive tract and maintain expression until stage 11 (Reim, 2003).
During stages 9 and 10, a new pattern of Doc expression emerges within dorsolateral areas of the germ band from parasegment (PS) 1 to PS13; the pattern consists of 13 rectangular cell clusters. This metameric expression includes ectodermal as well as underlying mesodermal cells. In the ectoderm, Doc expression is excluded from the dorsalmost cells near the amnioserosa at this stage, whereas in the ectoderm the metameric Doc expression extends to the dorsalmost areas of this germ layer. To determine the segmental register of Doc expression in the mesoderm, embryos were co-stained with antibodies against the POU domain transcription factor Cf1a (Ventral veins lacking/Drifter), which mark the tracheal placodes. Doc and Cf1a are expressed in mutually exclusive domains, which implies that Doc expression encompasses prospective tissues of the lateral epidermis and dorsolateral sensory organs. After stage 11, the segmental expression in the epidermis is modified to form segmental stripes that are interrupted in dorsolateral regions. Within these stripes, Doc expression is largely found in posterior areas of the anterior compartments of each segment and there is a graded distribution of Doc expression with increasing levels towards the posterior of each stripe. The dorsal epidermal expression domains now extend to the amnioserosa, and after dorsal closure the bilateral domains merge at the dorsal midline (Reim, 2003).
Additional sites of Doc expression during late embryogenesis include the dorsal pouch in the embryonic head, the anterior pair of Malpighian tubules and the pentascolopidial chordotonal sensory organs. The mesodermal expression of the Doc genes is observed in areas between the expression domains of the homeobox gene bagpipe (bap) at stage 10. This location defines them as dorsal areas of the mesodermal A (or slp) domains, which include the dorsal somatic and cardiogenic mesoderm. During early stage 11, additional Doc expression initiates in the caudal visceral mesoderm, which contains the founder cells of the longitudinal muscles of the midgut. Two out of six bilateral cardioblasts in each segment of the dorsal vessel (Lo, 2001), that are tin negative and svp positive, also express the Doc genes (Reim, 2003).
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).
The similarities in sequence and expression of the three Doc genes
suggests functional redundancy among these genes. Because molecular
analysis of available deficiencies at 66E-F shows that none of them uncovered all three genes, the flanking P-insertions EP(3)3556 and EP(3)584 were used in
attempts to delete the entire Doc gene cluster via male recombination-induced mutagenesis. Molecular mapping of the obtained
deletions demonstrates that two of them, Df(3L)DocA and
Df(3L)DocB, which were generated with the distally located insertion EP(3)3556 and caused embryonic lethality, delete all three Doc genes. Since
Df(3L)DocA deletes the smallest number of additional genes
(CG5087, CG5194, CG5144, Argk and CG4911), the
phenotypic analysis in the present study using this deficiency is described, although the salient phenotypes are very similar between Df(3L)DocA and Df(3L)DocB (Reim, 2003).
Additional genetic analysis shows that it is possible to obtain a small number of viable adult escapers with the genotype
Df(3L)Scf-R11/Df(3L)DocA, which indicates that
CG5087 is not absolutely required for viability, and that
Doc1 and Doc2 can functionally substitute for the loss of
Doc3. Similarly, the full viability of flies with the genotype
Df(3L)DocA/Df(3L)EP584MR2 shows that
CG4911 and the 5' exons of Argk (preceding the large
intron) are also not essential. Furthermore, it was determined that embryos with the genotypes Df(3L)Scf-R11/Df(3L)DocA and
Df(3L)DocA/Df(3L)29A6 (which causes pupal lethality) do not display any of the phenotypes described below for Df(3L)DocA
homozygous embryos. In summary, genetic analysis shows that the loss of
either Doc3 or Doc1 can be compensated for by the remaining two Doc genes in embryos and that the phenotypes are a
consequence of the loss of all three of the Doc genes. However, a contribution of CG5194, which encodes a 128 amino acid
predicted ORF with no known homology, to the observed phenotypes, cannot be ruled out (Reim, 2003).
Because of the prominent Doc expression in the primordia and developing
tissue of the amnioserosa, the amnioserosa marker Krüppel
(Kr) was used to examine whether the Doc genes are required for the
development of this extra-embryonic tissue. These experiments demonstrate
that homozygous Df(3L)DocA mutant embryos (henceforth called
DocA mutants) fail to express Kr in the amnioserosa at any
stage, whereas CNS expression of Kr is not affected. To confirm that this
observed phenotype is due to the loss of Doc gene function, Doc
gene functions were reduced by using RNA interference (RNAi) as an independent assay and
rescue experiments with DocA mutants were performed. Injection of a mixture of equimolar amounts of dsRNAs for all three Doc genes
frequently results in a complete absence of
Kr expression in the amnioserosa. The remaining embryos display
strongly reduced numbers of Kr-containing nuclei in this tissue. These phenotypes correlate with the observed absence or severe
reduction of Doc protein levels in Doc RNAi embryos. Hence, the strongest phenotype obtained by RNAi mimics the observed
DocA mutant phenotype, confirming that the lack of Kr
expression in DocA mutant embryos is specifically due to the loss of
the activity of all three Doc genes (Reim, 2003).
Besides the effects on Kr expression, DocA mutant and
RNAi-treated embryos share several morphological defects. The extending germ band is unable to displace the amnioserosa fully towards the anterior and the posterior germ band is therefore forced to bend underneath the amnioserosa. Of note, germ band retraction is strongly disrupted, which can be clearly seen in stage 14 embryos and
in cuticle preparations of unhatched first instar larvae. This phenotype is shared with previously described genes of the u-shaped
group, which affect the maintenance of the amnioserosa. Kr
expression and the germ band retraction defects in DocA mutant
embryos can be partially rescued by expressing any of the three Doc genes with an early amnioserosa-specific driver. Rescue with Doc2 is consistently more efficient when compared with Doc1 and Doc3, although it is not
known whether this difference is due to a higher intrinsic activity or a more efficient expression of Doc2 protein in this assay (Reim, 2003).
An additional phenotype consists of reductions in the size of the embryonic head in DocA mutants and RNAi-treated embryos, which is apparent from stage 12 onwards and results in reduced head structures and a frequent failure of head involution at later stages. This phenotype is probably due to excessive cell death as a consequence of the absence of Doc activity in the procephalic neuroectoderm and other dorsal areas of the embryonic head. The observed head phenotypes, as well as the aberrant shape of the filzkörper, are also reminiscent of similar phenotypes of embryos mutant for genes of the ush group (Reim, 2003).
To obtain more information about the particular role of the Doc genes in the specification and/or differentiation of the amnioserosa, the distribution of additional amnioserosa markers was analyzed in DocA mutant embryos. For the ush group gene hnt a
strong reduction of expression is found, with significant levels of Hnt protein being detected in nuclei only along the posterior margin of the amnioserosa. By contrast, the expression of the amnioserosa marker race (Ance) (is initiated normally in the primordium of the amnioserosa of DocA mutant embryos, suggesting that the expression of the race upstream activator zerknüllt (zen) is also not disrupted. However, after embryonic stage 9,
race expression is gradually lost in the amnioserosa of DocA mutant embryos and its residual mRNA distribution closely follows that of Hnt (Reim, 2003).
The expression of a novel amnioserosa marker, which is
encoded by the homeobox gene C15, was examined. In the normal situation, C15 is expressed in the amnioserosa from stage 7 until stage 17, when the amnioserosa undergoes apoptosis. In addition, from early stage 10 onwards there is a narrow
domain of expression at the leading edge of the dorsal germ band, which later becomes segmental. In DocA mutant embryos, the level of C15 expression in early amnioserosa cells is unaltered; this allows use of C15 protein as a marker for the development of this tissue in the absence of Doc activity (Reim, 2003).
Until stage 9, the large majority of amnioserosa nuclei in DocA mutant embryos appear large and flattened as in wild-type embryos. Together with data from alpha-tubulin staining, this observation indicates that the amnioserosa cells begin to acquire the normal features of a squamous epithelium. However, the amnioserosa does not display a properly folded morphology during stages 8-10, and the posterior germ band is forced to bend towards the inside in DocA mutant embryos. In addition, some small nuclei become detectable within the amnioserosa during this stage. Altogether, these observations indicate that the amnioserosa initiates its differentiation process in the absence of Doc gene activity but fails to complete it, thus leading to morphological and functional abnormalities of this tissue towards the end of germ band elongation. Much stronger alterations can be observed during subsequent stages, when there are an increasing number of C15-stained amnioserosa nuclei with much smaller diameters than regular amnioserosa nuclei. At late stage 12, almost all amnioserosa cells feature small nuclei that are difficult to distinguish from dorsal epidermal cells. Co-staining for race indicates that it is predominantly the cells with the small nuclei that lose race expression, while most normally-sized nuclei are still surrounded by race signals. From this stage onwards, non-stained 'holes' appear in the amnioserosa and the number of C15-stained amnioserosa nuclei decreases prematurely. Hence, unlike wild-type embryos, stage 14 DocA mutant embryos are not covered dorsally by C15-stained amnioserosa cells. In addition to the observed alterations in the amnioserosa, the C15 expression domain at the leading edge of the epidermis appears significantly broadened (Reim, 2003).
Is the increasing number of smaller nuclei in the
amnioserosa of DocA mutant embryos connected with abnormal cell
divisions? The M-phase marker phospho-Histone H3 can be detected in numerous amnioserosa nuclei of DocA mutant embryos after stage 10; this increase is not seen in wild-type embryos. In
addition, there is significant incorporation of BrdU in amnioserosa nuclei of DocA mutant embryos (particularly in the small nuclei, whereas no incorporation is observed in wild-type embryos. Mitotic spindles are also present in the amnioserosa of DocA mutants.
These observations indicate that the normal G2 arrest of amnioserosa cells has been released and the cells re-enter the cell cycle. Whether
the subsequent disappearance of small C15-stained amnioserosa nuclei in
DocA mutant embryos is a result of premature apoptosis of cells in this tissue was also tested. This possibility was confirmed by the results of TUNEL labeling experiments, which produced signals in many amnioserosa nuclei from 12 onwards. Most of the TUNEL-labeled nuclei have reduced or are lacking C15 expression, which shows that wild-type amnioserosa nuclei at late stage 12 are not apoptotic). Altogether, these observations suggest that loss of Doc activity prevents the normal differentiation of the amnioserosa to a fully functional tissue, suspends the cell cycle block of amnioserosa cells, and
causes premature apoptotic cell death in this tissue (Reim, 2003).
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date revised: 10 May 2006
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