The dorsal vessel forms from bilateral rows of cardioblasts and pericardial cells. The cardioblasts form a line of cells at the leading edge of the dorsal mesoderm. Pericardial cells are arranged in adjacent ventral rows. During dorsal closure, the rows of cardioblasts and associated pericardial cells migrate dorsally and the cardioblasts fuse with their contralateral partners. The two rows of cardioblasts are located medially and fuse to form the lumen of the dorsal vessel and are flanked by rows of noncontractile pericardial cells located ventrally, laterally, and dorsolaterally. H15 (Brook, 1996) and mid (Buescher, 2004) are linked T-box genes that share 89% identity in their T-box domains. The H15-lacZ enhancer-trap (Brook, 1993), inserted 3 kb 5' of H15, shares the expression pattern of both mid and H15 during ectodermal segmentation (Buescher, 2004). Griffin (2000) reported that H15-acZ was expressed in cardioblasts. The mid and H15 expressions in the embryo are first detected in ventral ectodermal stripes and neuroblasts (Buescher, 2004). In the mesoderm, mid is first expressed during early stage 12, where it is detected in two cells per hemisegment in second thoracic through eighth abdominal segments (T2-A8). During stages 12 and 13, the expression pattern evolves into clusters of cells and then into a continuous row of cells in the dorsal mesoderm that fuse dorsally. H15 mRNA is first detected in the dorsal mesoderm in clusters of cells at stage 13, after which H15 is expressed in the same pattern as mid but at lower levels. To confirm that mid and H15 are expressed in cardioblasts, mid and H15 mRNA were examined in embryos carrying an enhancer trap inserted at the seven-up locus (svp-lacZ). svp is expressed in two cardioblasts per hemisegment, and both mid and H15 mRNA colocalize with svp-acZ. As predicted for cardioblast-specific expression, each pair of mid and H15 svp-lacZ-expressing cells is separated by four cells expressing only mid and H15. Thus, the mid and H15 mRNAs are localized to cardioblasts in the mature dorsal vessel (Miskolczi-McCallum, 2005).
Unlike tin and pnr, which are expressed in broad domains throughout the dorsal mesoderm before becoming restricted to a subset of heart cells, mid is initially activated in two cells per hemisegment. To determine where the cells arise, the location of mid-expressing cells was examined in the dorsal mesoderm relative to the ectodermal expression of the segmental markers Wg and Engrailed (En). Wg and En mark adjacent domains in ectoderm overlying the dorsal mesoderm with Wg labeling the row of cells just anterior to the En domain. The two mid-expressing cells arise in similar positions in the underlying mesoderm in each hemisegment. One cell located beneath the Wg stripe and the other lying beneath and slightly to the posterior of the En stripe. As stage 12 progresses, groups of three to six mid-expressing cells are located beneath or near the Wg and En stripes, consistent with the two initial cells dividing and coalescing to form clusters. The clusters fuse to form a continuous line of cells with six cells per hemisegment. The progression of mid expression is consistent with previous mosaic analysis and experiments manipulating the cell cycle in the dorsal mesoderm cells. These studies provided indirect evidence that the six cardioblasts in each hemisegment arise from two progenitors that divide twice to give rise to all six cardioblasts and two of the pericardial cells (Miskolczi-McCallum, 2005).
Previous work has shown that loss of Notch signaling results in a fourfold increase in cardioblasts. To test whether the expression of mid is dependent on the Notch pathway, Notch signaling was blocked with a loss-of-function mutation in the ligand Delta (Dl). Seven to ten mid-expressing cells were observed in each hemisegment in early stage 12 Dlx mutant embryos. Each cluster of mid-expressing cells is associated with an ectodermal En stripe, although clusters are occasionally missing or fused. To ectopically activate the Notch pathway, the intracellular domain of Notch (Nicd) was expressed throughout the mesoderm using the GAL4/UAS system. In twi-GAL4;UAS-Nicd embryos, mid expression in the dorsal mesoderm of stage 12 embryos was often absent. These results suggest that the mid-expressing cardioblast precursors are selected from a single pool of competent progenitors by Notch-mediated lateral inhibition (Miskolczi-McCallum, 2005).
Given the documented pattern of vertebrate Tbx20 expression in heart, expression of Drosophila Tbx20/neuromancer (nmr) was examined in the forming heart. Using fluorescent in situ hybridization, it was determined that both nmr1 (H15) and nmr2 (midline) genes exhibit similar RNA expression patterns late in embryogenesis, but they differ significantly in the onset of expression. Cardiac nmr2 expression initiates at late stage 11 when tinman is restricted to the prospective cardiac mesoderm, whereas nmr1 RNA is only detectable after stage 13. Both persist in an identical pattern as the bilateral myocardial cells begin to assemble into the heart tube at the dorsal midline. nmr2 is first detected at blastoderm stage as well as later in ventral ectoderm derivatives posterior to Wg and overlapping with En/Hh (Qian, 2005).
Since nmr2 is not expressed in the entire tinman-expressing cardiogenic region, fluorescent in situ hybridization of nmr2 double-labeled with various markers of other sets of cardiac progenitors was conducted. Indeed, nmr2 is expressed in a complementary pattern to that of Eve, which marks a subset of pericardial cells. After germ band retraction, the nmr2-expressing cells differentiate into 6 myocardial cells per hemisegment indicated by double labeling with nmrH15-LacZ. The exclusive expression of nmr2 (and nmr1) in all myocardial cells that will give rise to the contractile myocardium suggests that the corresponding cardiac progenitors may co-label with Lbe and Svp, in addition to Tinman. Indeed, double-labeling with Lbe shows that they coincide in two myocardial cells per hemisegment. Cardiac nmr expression (after stage 13) also coincides with other myocardial-specific (e.g., dSUR) but not with pericardial-specific markers (e.g., Zfh-1). However, the possibility cannot be excluded that at earlier stages (11/12) mixed or pericardial-only lineages (e.g., expressing tinman but not eve) transiently express nmr (Qian, 2005).
During germ-band extension, Dpp signals from the dorsal ectoderm to maintain Tinman (Tin) expression in the underlying mesoderm. This signal specifies the cardiac field, and homologous genes (BMP2/4 and Nkx2.5) perform this function in mammals. A second Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1. Via Zfh1, the second Dpp signal restricts the number of Odd-skipped-expressing and the number of Tin-expressing pericardial cells. Dpp also represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number. In the adjacent dorsal muscles, Dpp has the opposite effect. Dpp maintains Krüppel and Even-skipped expression required for muscle development. The data show that Dpp refines the cardiac field by limiting the number of pericardial cells. This maintains the boundary between pericardial and dorsal muscle cells and defines the size of the heart. In the absence of the second Dpp signal, pericardial cells overgrow and this significantly reduces larval cardiac output. This study suggests the existence of a second round of BMP signaling in mammalian heart development and that perhaps defects in this signal play a role in congenital heart defects (Johnson, 2007).
A previous study suggested that a second round of Dpp dorsal ectoderm-to-mesoderm signaling, stimulated by enhancers located in the dpp disk region, initiates during germ-band retraction (stage 12; Johnson, 2003). This is referred to as the second round of signaling because a distinct set of enhancers located in the dpp Haplo-insufficiency (Hin) region activates Dpp dorsal ectoderm-to-mesoderm signaling during germ-band extension (stage 8). Further, the data revealed that dpp dorsal ectoderm expression driven by the Hin region enhancers persists long after germ-band retraction. These studies showed that Hin-region-driven dpp expression is sufficient for Dpp ectodermal functions such as dorsal closure and dorsal branch migration (Johnson, 2007).
Given these data, it appears that the dppd6 inversion prevents the augmentation of dpp expression in the dorsal ectoderm during germ-band retraction that is normally provided by disk region enhancers. The presence of numerous mesodermal phenotypes in dppd6 mutants (Johnson, 2003) suggests that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals so that they can reach the underlying mesoderm. Perhaps there are barriers of distance or extracellular matrix density between these germ layers that must be overcome (Johnson, 2007).
The data are wholly consistent with the hypothesis that the dppd6 inversion prevents the augmentation of dpp expression provided by disk region enhancers during germ-band retraction. The data further suggest that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals such that they can reach the underlying mesoderm. Finally, this study shown that during germ-band retraction Dpp signals maintain the boundary between pericardial cells and dorsal muscle cells via two distinct mechanisms: the regulation of gene expression and the restriction of cell proliferation. To regulate gene expression, Dpp signals directly to pericardial cells and restricts Odd and Tin expression in a zfh1-dependent manner. Dpp also limits Tin expression, independently of zfh1, by repressing the expression of mid, a stimulator of proliferation (Johnson, 2007).
With respect to zfh1-dependent regulation, the data support the hypothesis that Dpp restricts Zfh1 expression to regulate the number of pericardial cells derived solely from symmetrically dividing lineages. Lineage analyses have identified both symmetric and asymmetric cell divisions of myogenic and pericardial precursor cells. Pericardial cells are derived from four separate lineages that arise from four distinct precursor cells. Asymmetric precursor cell divisions initiating between stages 8 and 10 give rise to the Odd-positive/Seven up (Svp)-positive pericardial cells and the Eve-positive/Tin-positive pericardial cells (EPCs). In contrast, symmetric division, initiating at the same stage, establishes the Odd-positive/Svp-negative pericardial cells (OPCs) and the Tin-positive/Eve-negative pericardial cells (TPCs). dpp mutations do not affect the number of EPCs or the number of Odd-positive/Svp-positive cells. However, embryos bearing dpp mutations show an increase in the number of OPCs and TPCs. Therefore, the ectopic pericardial cells seen in dpp mutants derive from symmetrically dividing lineages (Johnson, 2007).
Previous reports have shown that regulation of asymmetric cell division is a key mechanism in establishing boundaries among the various cell types in the dorsal mesoderm. For instance, in the absence of Numb, a Notch pathway antagonist, asymmetric progenitor cell division is abrogated and the number of Odd-positive/Svp-positive cells and EPCs increases at the expense of the Svp-expressing cardial cells and Eve-expressing dorsal muscle cells, respectively. This study extends these observations by showing that pericardial cell types derived from symmetrically dividing lineages are also under strict regulatory control (Johnson, 2007).
With respect to zfh1-dependent regulation of pericardial cell number, Dpp restricts cell proliferation and, in turn, Tin expression by limiting mid expression. In wild-type embryos, cell division in the dorsal mesoderm is largely complete by the early stages of germ-band retraction (stage 11), whereas in dppd6 embryos cell proliferation in the dorsal mesoderm continues through stage 13. Interestingly, the number of cells expressing Zfh1 increases from stage 12 to stage 13 in wild-type embryos in the absence of cell division, demonstrating that patterning events subsequent to cell division regulate cell fate choices in the dorsal mesoderm. This hypothesis is supported by the fact that tracing pericardial cell lineages requires inducing mitotic clones by stage 8. Therefore, the ectopically dividing mesoderm cells observed in dppd6 embryos are derived from cells with the potential to become Tin-expressing cells (Johnson, 2007).
During stage 12, tin expression is reactivated in a subset of cardiac cells in a mid-dependent fashion, suggesting that tin expression in precursor cells alone is not sufficient for specifying the ultimate fate of their daughter cells. Moreover, misexpression of mid results in both ectopic cell division and expanded tin expression. Lineage studies support the necessity of reactivating Tin by showing that a single precursor cell gives rise to two Tin-positive/Eve-negative pericardial cells and two siblings that do not express Tin. Thus tin is not reactivated in all subpopulations of pericardial cells. The data suggest that, during stage 12, Dpp prevents tin reactivation in cells occupying lateral regions of the dorsal mesoderm by limiting mid expression (Johnson, 2007).
Development of the dorsal musculature initiates when founder cells are specified in the mesoderm. These founder cells then fuse with neighboring cells to form syncitial myofibers. In the absence of Dpp, the pericardial cell domain expands into the dorsal muscle domain and reduces expression from the dorsal muscle genes Kr and Eve. Since the separation between pericardial and dorsal muscle cells is lost in dpp mutant embryos, it is concluded that Dpp maintains the pericardial-dorsal muscle cell boundary after it is established. Moreover, reducing pericardial cell number increases Kr expression after germ-band retraction, suggesting that cross-repressive interactions between pericardial and dorsal muscle cells contribute to patterning of the dorsal mesoderm. The presence of ectopic pericardial cells in the dorsal mesoderm reduces the number of myofibers comprising the dorsal muscles even though the dorsal muscle founder cells are, for the most part, correctly specified. pMad does not accumulate in Kr-expressing founder cells yet Kr expression is significantly reduced in dpp mutant embryos. Therefore, changes in Kr and Eve expression observed in embryos with altered dpp or zfh1 activity reflect alterations in the number of myoblast fusion events in the dorsal mesoderm (Johnson, 2007).
These data extend a previous study showing that misexpressing Zfh1 reduces dMef2 expression in somatic muscles. This study demonstrates that misexpression of Zfh1 induces ectopic pericardial cells and that the presence of pericardial cells in the dorsal muscle domain reduces myoblast fusion. Therefore, reduced dMef2 expression in embryos misexpressing Zfh1 is likely the result of reduced myoblast fusion and not of direct repression of dMef2 expression by Zfh1. Further, analysis of lmd mutants that have reduced numbers of myoblasts revealed that they also contain an excessive number of pericardial cells. Together, these results suggest that maintaining the pericardial-dorsal muscle cell boundary requires Dpp-mediated cross-repressive interactions between these cell types. Thus, in the absence of Dpp, the transformation of dorsal muscle cells into pericardial cells reduces the number of myoblasts available for fusion (Johnson, 2007).
Experiments in the larvae of Drosophila and other insects suggested that pericardial cells act as nephrocytes that filter the hemolymph. These studies also showed that pericardial cells secrete proteins into the hemolymph, suggesting that one pericardial cell function may be to provide short- or long-range signals. Consistent with this, reducing pericardial cell number reduces heart rate and increases the cardiac failure rate, suggesting that pericardial cells influence the development of cardiac cells (Johnson, 2007).
This study shows that pericardial cell hyperplasia reduces the luminal distance of the heart during systole as well as diastole, resulting in an overall decrease in average pulse distance of each contraction. However, pericardial overgrowth does not alter heart rate, indicating that cardiac cells develop appropriately in the presence of ectopic pericardial cells. Luminal measurements suggest a role for pericardial cells in the mechanics of heart function. One hypothesis for this is based on the fact that pericardial cell hyperplasia results in excess levels of extracellular matrix protein Pericardin (Prc) in the extracellular matrix (ECM) surrounding the heart. Prc is a collagen IV-like ECM protein secreted at high levels from pericardial cells. In dpp mutants, excess Prc is seen predominantly in the posterior of the heart where the pulse-distance reduction was observed. It is proposed that Prc secreted by pericardial cells limits the width of the dorsal vessel at diastole and thus modulates the pulse distance of each heart contraction. Pericardial cell overgrowth would increase Prc deposition, thereby reducing the size of the diastolic heart and the pulse distance. Consistent with this hypothesis, excessive expression of ECM proteins, including collagen IV, was correlated with heart failure in patients presenting with end-stage cardiomyopathy (Johnson, 2007).
It is well documented that many of the early events driving Drosophila embryonic heart development have been conserved in vertebrates. The data provide the first basis upon which to determine if Dpp regulation of Zfh1 or Tin late in heart development is also conserved (Johnson, 2007).
Two orthologs of zfh1, Sip1 and Kheper, have been identified in vertebrates. Zebrafish embryos injected with the Dpp homolog BMP4 show reduced Kheper expression while Xenopus embryos injected with the BMP antagonist Chordin display elevated Sip1 expression. These results suggest the possibility that Dpp repression of zfh1 expression may be conserved in vertebrates. In addition, mammalian Sip1 plays an essential role in heart development. In mice, Sip1 is expressed in neural crest cells (NCCs), paraxial mesoderm, and neuroectoderm. The subset of NCCs that express Sip1 give rise to the septum and large arteries of the heart. Sip1 knockout mice fail to form these NCCs and these mice die midway through gestation with numerous heart defects. Mice lacking the BMP receptors BMPRIA or ALK2 specifically in NCCs also display numerous cardiac phenotypes. In conditional knockout of ALK2 in NCCs, abnormalities are seen in the heart's outflow tract, and conditional knockout of BMPRIA in NCCs results in heart failure and early embryonic lethality similar to Sip1 knockout mice. Thus BMP signals are required for proper specification of NCCs, and loss of BMP signaling in NCCs phenocopies Sip1 knockout mice to an extent. It is tempting to speculate that, as in Drosophila, BMP signals regulate the Zfh1 ortholog Sip1 to correctly specify NCCs and, in turn, to properly pattern the mammalian heart (Johnson, 2007).
With regard to the conservation of late-stage Dpp regulation of Tin, a recent article describing a study of mice with a conditional knockout of Nkx2.5 where expression is missing only during late stages of heart development (post E14.5) is highly relevant. Utilizing rescue of Nkx2.5 mutant embryos with BMP-signaling-pathway components, the study identified a direct connection among BMP4 signaling, Nkx2.5 activity, and heart cell proliferation. Since Nkx2.5 is the Tin homolog, BMP4 is the Dpp homolog, and the mutant phenotype (heart cell hyperplasia) is the same in both species, this suggests that this aspect of Dpp signaling is conserved in mammals. Together with this study, these results suggest that defects in late-stage BMP signaling may play a role in congenital heart defects (Johnson, 2007).
Because mid and H15 expression coincides with cardioblast fate, tests were performed to see whether mid and H15 are required for cardioblast formation and dorsal vessel development. Because the two genes are very closely linked, a pair of overlapping deficiencies was used to generate embryos deleted for both mid and H15. Df(2L)GpdhA deletes genes beginning with H15 moving proximally, and Df(2L)x528 deletes genes beginning with mid and moving distally (Buescher, 2004). Df(2L)x528/Df(2L)GpdhA (H15 mid null) animals are embryonic lethal and have ventral segmentation defects due to the early essential role that has previously been demonstrated. It is important to note that the expression and effects of mid and H15 in segmentation are restricted to the ventral ectoderm (Buescher, 2004) and thus the loss of mid and H15 does not affect the segment polarity gene expression in the dorsal ectoderm that is required for the proper induction of heart precursors (Buescher, 2004). The region deleted by both deficiencies contains only H15, midline, and a predicted gene of unknown function, CG31467. Embryos were probed with CG31467 anti-sense RNA probes and no hybridization was detected and thus CG31467 is unlikely to contribute to dorsal vessel development (Miskolczi-McCallum, 2005).
To characterize the effects of mid and H15 loss of function on heart development, mutant and control embryos were labeled with antibodies to Tinman. At the end of morphogenesis, each cardioblast pairs with a cardioblast in the opposite row to form the central tube of the dorsal vessel. Four out of six cardioblasts pairs in each segment are labeled with Tin and these can be seen as ordered groups of four pairs of labeled cells separated by two pairs of unlabelled cells. Rows of pericardial cells, marked with Zfh-1 or Zfh-1 and Tin, flank the central rows of cardioblasts. In virtually all H15 mid null embryos, the pairing of the Tinman+ cardioblasts is abnormal with some of the cells pairing and others not in proper alignment. In some cases, the central tube of the dorsal vessel is twisted with one row of cardioblasts lying beneath the other. Pericardial cells are often displaced either laterally or medially from their normal location, possibly as a secondary consequence of defects in cardioblast alignment. The overall disorganization is most severe in the middle of the dorsal vessel, in abdominal segments 3, 4, and 5 (Miskolczi-McCallum, 2005).
In order to test the individual contribution of mid and H15 to the dorsal vessel phenotype, embryos mutant for one gene but not the other were examined. H15x4/Df(2L)GpdhA embryos are deleted for both copies of H15 and one copy of mid (H15 null) and showed no heart defects. In contrast, embryos homozygous for the null mutation mid1 (Buescher, 2004; Nüsslein-Volhard, 1984) have a variable heart phenotype. Typically, 50%-60% of mid1 homozygous embryos exhibit an overall disorganization of the dorsal vessel as indicated by Zfh-1 and Tin double labeling while the rest have fairly normal heart morphology. The phenotypes are similar to but weaker than the phenotype seen in H15 mid null embryos. These results suggest that mid and H15 play critical, partially redundant roles in the morphogenesis of the dorsal vessel with mid having a greater role but with loss of H15 increasing the penetrance and expressivity of the defects. A similar relative requirement has been demonstrated (Buescher, 2004) for mid and H15 in ectoderm segmentation (Miskolczi-McCallum, 2005).
To test whether mid and H15 were required for cell fate specification in the dorsal vessel, the expression of several cardioblast and pericardial cell markers was examined in both wild-type embryos and in H15 mid null embryos. DMef2 encodes a MADS box transcription factor expressed in most muscle cells and is specific to cardioblasts in the heart. In wild-type embryos, Dmef2 marks 52 cardioblasts in each half of the heart. In H15 mid null embryos, the wild-type number of Dmef2 cells is found (51.8, n = 11), indicating that cardioblasts are properly formed in the absence of mid and H15 function. The expression of B2-3-20, a lacZ enhancer trap that marks cardioblasts, and Myosin Heavy Chain (MHC), a marker of muscle differentiation, were also found to be normal in H15 mid null embryos, as was the expression of the Hand mRNA, a marker of all cardioblasts and pericardial cells (Kolsch, 2002). These results suggest that mid and H15 are not essential for cardioblast specification. Pericardial cells were also examined in wild-type and H15 mid null embryos. Zfh-1 labeling was counted in confocal slices from mutant and wild-type embryos to estimate pericardial cell numbers. In segments A2-A8, which are posterior to the lymph gland, similar numbers of Zfh-1-positive cells were found in wild-type (118.5, n = 7) and H15 mid null embryos (121.5, n = 9). The staining of Eve, a marker of two pericardial cells per hemisegment, was also normal in H15 mid null embryos, suggesting that mid and H15 are also not required for the generation of correct numbers of pericardial cells. One difference was noted in the lymph gland, a structure composed of Zfh-1-labeled cells that surround the two anterior-most segments of the dorsal vessel, T3 and A1. An increase in Zfh-1-positive cells was found, with 63.7 cells labeled per gland in H15 mid null embryos compared to 50.9 cells labeled per gland in wild-type embryos. Because mid and H15 are not expressed in the lymph gland primordia, this may be a nonautonomous effect of mid and H15 loss of function in the dorsal vessel (Miskolczi-McCallum, 2005).
The loss-of-function data indicate that mid and H15 are required for heart morphogenesis but are not essential for the specification of cardiac fate. In order to assess the effect of ectopic mid expression on cardiac development, mid was expressed with a UAS-mid construct (Buescher, 2004). Expressing UAS-mid under the control of twist-GAL4 drives mid throughout the mesoderm beginning in gastrulation. An increase in Tin-positive cells was detected in the dorsal mesoderm of twi-GAL4;UAS-mid embryos. By stage 13, a clear increase in the number of Tin-positive cells is visible, suggesting an increase in cardiac fate promoted by the ubiquitous expression of mid in the mesoderm. Similar effects were seen using pnr-GAL4, which drives GAL4 in dorsal mesoderm and dorsal ectoderm, indicating that ectopic mid expression in dorsal mesoderm is sufficient to cause the observed effects (Miskolczi-McCallum, 2005).
The dramatic increase of Tin-positive cells caused by ectopic mid expression in the dorsal mesoderm led to an examination of the effects of ectopic mid expression on visceral mesoderm development, which is also derived from dorsal mesoderm. The expression of Fasciclin III (Fas III), a marker of visceral mesoderm was examined in twist-GAL4;UAS-mid embryos. In many embryos, reduced or gapped expression of Fas III was found, consistent with a loss of visceral mesoderm as a result of ectopic mid expression (Miskolczi-McCallum, 2005).
In addition to the changes in tin and Fas III expression, ectopic mid expression driven by pnr-GAL4 or twi-GAL4 disrupts heart morphogenesis, often preventing the two halves of the heart from fusing and making it difficult to assess how ectopic mid affects specific cardiac lineages. However, when expressing UAS-mid under the control of How-GAL4, another driver with expression throughout mesoderm, similar but weaker effects on Tin and Fas III distribution was observed in stage 13 embryos compared to those observed for twist-GAL4;UAS-mid embryos. Furthermore, a range of defects is seen in older embryos. In some embryos, there is a disruption of heart development similar to that seen with twi-GAL4 and pnr-GAL4, but in most embryos, the two halves of the heart join together but have lateral branches and ectopic cells. The lateral branches contain Tin-positive cells, Zfh-1-positive cells, and Tin-positive Zfh-1-positive cells. The ectopic Zfh-1-positive cells and Tin-positive Zfh-1-positive cells likely represent ectopic pericardial cells. The ectopic Tin-positive cells are likely to be ectopic cardioblasts. To confirm this, β-galactosidase-labeled cardioblasts were examined in How-GAL4;UAS-mid embryos carrying the H15-lacZ enhancer-trap. Ectopic cells labeled with β-galactosidase were observed in branched patterns. In normal H15-lacZ embryos, 52 β-Gal-positive cardioblasts per side were found in the dorsal vessel. In H15-lacZ;How-Gal4;UAS-mid embryos, up to 30 extra cardioblasts were found per half dorsal vessel. Double labeling with Dmef2 further supports the identity of the ectopic cells as cardioblasts. In summary, the loss-of-function data indicate that mid and H15 are not required for fate specification, while the gain-of-function data indicate that ectopic expression of mid in dorsal mesoderm is sufficient to induce ectopic cardiac fate (Miskolczi-McCallum, 2005).
To determine the requirement for nmr in Drosophila heart formation, mutants were generated. The nmr1 mutants, nmr1614 and nmr1210, were made by imprecise excision of the H15 P-element located 331 bp 5′ to the transcript start. In both alleles, the 5′UTR, transcription and translation start and part of T-box domain are deleted. In both alleles, no nmr1 transcripts are detected as determined by RT-PCR and whole-mount in situ hybridization, but nmr2 RNA appears unchanged (Qian, 2005).
In order to reduce or eliminate nmr2 function, a transgenic snapback RNA interference (RNAi) approach was taken in conjunction with the UAS-Gal4 system. The cDNA fragment of nmr2 with least homology to nmr1 was used to generate transgenic flies containing UAS-nmr2RNAi. When da-Gal4 is driving nmr2-RNAi ubiquitously, nmr2 RNA is virtually absent. By combining nmr2RNAi transgenes with nmr1 mutants, a quasi-allelic series of loss-of-function was generated for both nmr genes. To distinguish the mesodermal versus ectodermal requirement of nmr, germlayer-specific nmr double mutants were generated. For example, embryos of the genotype nmr1614; 24B-Gal4/UAS-nmr2RNAi (nmr1−,nmr2meso−) lack nmr2 RNA in the heart region (Qian, 2005).
nmr1 mutants are viable and exhibit only mild defects in cardiac morphogenesis, and none of the early heart markers seem to be affected. In twi24B-Gal4/UAS-nmr2RNAi (nmr2meso−) embryos, however, eve expression is moderately expanded. Eliminating both nmr genes in the mesoderm (nmr1−,nmr2meso−), a more severe phenotype is observed, in that at stage 12, the Eve clusters are dramatically enlarged. Importantly, this expansion appears to be at the expense of lbe expression, which is significantly reduced. Despite the change in the relative proportion of eve and lbe expression in almost half of the double mutant embryos, the extent of cardiac mesoderm delineated by tinman expression is not appreciably altered at this stage. Visceral mesodermal markers also do not seem to be noticeably affected (Qian, 2005).
Consistent with the increase in Eve cell formation with reduced nmr function is the complementary pattern of nmr2 expression relative to Eve positive cells. This raises the possibility that nmr normally antagonizes eve expression, similar to the repression of eve by lbe. In order to test this, nmr was ectopically expressed to see if this causes a reduction in eve expression. Indeed, overexpression of either nmr throughout the mesoderm greatly reduces stage 12 eve expression while expanding lbe. This repression may not (only) be direct, but may (also) be mediated via activation of lbe, for example, which then directly represses eve. The repressive activity of Nmr is consistent with in vitro mouse cell culture studies, in which Tbx20 acts as a repressor rather than an activator (Plageman, 2004). Interestingly, eve is not only repressed by nmr, but eve itself can repress nmr, again consistent with the cross-repressive relationship between eve and lbe. Therefore, it is proposed that nmr plays a crucial role in the distinction of cardiac cell types during the early events of cardiogenesis (Qian, 2005).
After germ band retraction (stage 13/14), the cardiac progenitor lineages have produced a set of contractile myocardial and pericardial cells that will go on to form the mature heart. Since at that stage nmr expression has become restricted to all prospective myocardial cells, their differentiation was examined in nmr mutants, using myocardial- and pericardial-specific markers. Remarkably, even though cardiac-restricted Tinman is not significantly altered at stage 12, by stage 14 myocardial Tinman is dramatically reduced in nmr1−,nmr2meso− embryos. The remaining Tinman-positive nuclei often co-label with Eve and are thus of pericardial identity. In addition, staining with muscle-specific transcription factor Dmef2 (normally present in all myocardial but none of the pericardial nuclei) is less or absent in the heart-forming region in these nmr mutant embryos. It is speculated that the reduction in tinman and Dmef2 expression in the forming myocardium is in part due to the loss of lbe expression, which includes the Lbe myocardial progenitors co-expressing tinman. To account for the loss of non-Lbe positive myocardial cells, it is postulated that the Lbe-negative, Tinman-positive myocardial progenitor lineages also require nmr function. Since at stage 12 most if not all cardiac progenitors express tinman and their pericardial and myocardial progeny are not yet distinguishable by position, tinman expression appears normal. It is only later when the lack of myocardial differentiation becomes apparent (Qian, 2005).
Even though the non-myocardial Eve progenitor clusters are enlarged in nmr mutants, the number of progeny Eve pericardial cells (EPCs) is only moderately increased. Another marker of pericardial cells, Odd-skipped (Odd), which does not overlap with Eve, was examined. Indeed, odd expression is also expanded in nmr1−,nmr2meso−. This suggests that without nmr function pericardial cell populations are expanded, whereas myocardial differentiation is compromised. Conversely, mesodermal overexpression of nmr diminishes Eve and Odd pericardial cell fates, but increases the number of Dmef2 myocardial cells (Qian, 2005).
Since pannier is able to induce transient ectopic tinman expression in the mesoderm, it was asked if nmr also has that capacity. Indeed, mesodermal expression of either nmr gene causes abundant ectopic tinman expression until about stage 14, but does not persist later. This is consistent with the idea that one of nmr's role is to cooperate with tinman (and pannier, see below) in determining the dorsal-most mesoderm as cardiogenic (Qian, 2005).
The dramatic nmr-induced ectopic tinman expression may also be due to increased proliferation of cardiac progenitors. In vertebrates, for example, it has been shown that Tbx5 can inhibit cell proliferation in vitro. Moreover, loss-of-Tbx20-function in mice results in hypoplastic hearts because of reduced cardiac proliferation. To determine whether nmr affects proliferation in Drosophila, the mitotic marker phospho-histone H3 (pH3) was used. While wildtype embryos exhibit only few proliferating cells in the dorsal mesoderm at stage 13, pan-mesodermal nmr expression increases the number of pH3-labeled nuclei throughout the mesoderm, including the cardiogenic region. At earlier stages (late 11/12), proliferation is also increased compared to wildtype, suggesting that nmr is capable of promoting mesoderm proliferation, in addition to its clear role in cardiac lineage specification. Therefore, it is possible that the loss of myocardial cells in nmr mutants is not only due to misspecification, but because nmr may also be required for myocardial progenitor proliferation. Although the ventral mesoderm also shows excess nmr-induced proliferation, it is unlikely, however, that the cells in that region expressing ectopic tinman originate from over-proliferating (dorsal) myocardial progenitor, since there is no gradient of ventrally migrating cells observed. In order to conclusively address this point, the nmr overexpression phenotype will have to be examined in cell cycle arrest mutants (Qian, 2005).
During the process of dorsal closure, the bilateral primordia of the heart migrate towards the dorsal midline and acquire a polarity that is the result of a typical mesenchyme-epithelium transition. This study has explored whether the morphogenetic mechanism of coordinate myocardial cell alignment and heart tube assembly is controlled by nmr. A large proportion of nmr2meso− or nmr1−,nmr2meso− embryos at stage 16/17 exhibit considerable myocardial misalignment defects. This phenotype includes intercalation, gaps, clustering and mis-orientation of the Dmef2-labeled myocardial cells (Qian, 2005).
To explore the cellular mechanism of how nmr regulates myocardial cell alignment, the cytoskeletal architecture and epithelial polarity features of the forming myocardium were monitored. First, the cardiac expression of epithelial polarity markers, namely discs large (dlg), armadillo (arm), crumbs (crb), dmPar6, dystroglycan (dg), α-spectrin and βH-spectrin were examined. Among them, dlg, arm, dg and α-spectrin are expressed in the heart in a stereotyped localized pattern (Qian, 2005).
dlg encodes a MAGUK protein containing three PDZ domains and is required for apical-basal polarity by localizing to the basal-lateral sides of epithelial cells. In myocardial cells of the forming heart tube, however, Dlg protein seems to localize to the dorsal and lateral sides of these cells. Even though the leading edge of the dorsally migrating bilateral rows of prospective myocardial cells may be thought of as 'apical', since they meet at the dorsal midline, the 'new' basal side of this forming heart epithelium is facing the midline and prospective lumen of the heart tube, as is the case for blood vessels in vertebrates. In any case, Dlg is severely mislocalized in myocardial cells of nmr mutants (Qian, 2005).
α-Spectrin also marks the basal-lateral membrane of epithelial cells, and in Drosophila is detected at the dorsal and lateral sides of the myocardium, similar to Dlg. In nmr mutants, α-Spectrin is also mislocalized in the forming heart; but unlike Dlg, α-Spectrin levels are much reduced and frequently non-detectable. The similar phenotype is observed with Arm (Qian, 2005).
Dystroglycan (Dg), a major component of the Dystrophin-Glycoprotein complex, plays a crucial role in the establishment of epithelial polarity of the follicle cells in the Drosophila ovary. Dg strongly accumulates at both the basal and apical sides of myocardial membranes, but is excluded laterally. As with the other polarity markers, Dg localization is dramatically disrupted in nmr mutant embryos. The mislocalization of these polarity genes indicates a failure of the myocardium in proper polarity acquisition. Thus, in addition to their role in cell fate specification, the nmr encoded Tbx20 genes may also coordinate the mesenchyme-epithelium transition and polarized alignment of the myocardial primordia (Qian, 2005).
Since loss-of-nmr-function results in early cardiac specification defects, it is possible that the observed abnormalities in morphogenesis are secondary effects to the early defects. Thus, it was of interest to determine whether or not the early misspecifications due to loss-of-nmr-function can be separated from the later morphogenesis defects. Instead of expressing nmr2RNAi with the pan-mesodermal driver 24B-Gal4, the heart-specific tinCΔ4-Gal4 was used; this drives expression in differentiating myocardial cells as they migrate towards the dorsal midline was used. In tinCΔ4 > nmr2RNAi embryos, early cardiac specification does not seem to be affected, based on Tinman, Eve and Dmef2 staining, nor is dorsal migration or alignment of myocardial cells at the dorsal midline. In contrast, however, epithelial polarity markers are often mis-localized as determined by Dlg, α-Spectrin and Dg staining. Dlg, for example, is no longer detected on the dorsal-lateral sides, but instead is frequently deposited also ventrally or missing on the dorsal myocardial membranes. These data suggest that nmr is required for myocardial cell polarization independently of its role in cell fate specification (Qian, 2005).
The GATA transcription factor Pannier has been shown to be required, along with Tinman, for specification of the heart primordium. pannier null mutant embryos exhibit a reduction of both myocardial and pericardial cell populations, but eve expression is less affected than that of lbe. Thus, the functional relationship between pannier and nmr in cardiac cell type specification was examined. Double-labeling for pannier RNA and nmrH15-LacZ shows that reporter gene expression overlaps with that of pannier at the dorsal edge of the mesoderm. In pannier null mutants, mesodermal nmr expression is completely missing, which is in contrast to the presence of residual tinman and other cardiac marker genes. In addition, pan-mesodermal expression of pannier is sufficient to initiate nmr expression ectopically. In contrast, misexpression of nmr throughout the mesoderm is unable to induce pnr ectopically, and in nmr1−,nmr2meso− embryos no detectable change in pannier expression is observed. These findings are consistent with the idea that nmr acts downstream of pannier (Qian, 2005).
To further examine the relationship between nmr and pannier, whether they interact genetically was examined. In pannier mutants, the Eve clusters are moderately reduced, whereas in nmr mutants an increase in the size of these clusters is observed. In contrast, a further Eve cluster reduction is observed in nmr1;pnr double mutants. A similar synergistic enhancement of the pannier phenotype by nmr1 is observed using Tinman as a marker. This is consistent with in vitro data that show a direct interaction between Tbx and Gata factors. However, this also raises the question why nmr1− interacts synergistically with nmr2meso− to enlarge the Eve clusters, whereas with pannier− a further reduction of Eve is observed. It is thus possible that during cardiac mesoderm specification nmr not only plays an essential role in the distinction between Eve and Lbe progenitors, but also acts (redundantly) in conjunction with pannier to maintain tinman expression, which in turn is required for maintaining eve expression. In agreement with this interpretation is the observation that nmr overexpression can induce tinman ectopically (Qian, 2005).
Whether nmr expression is regulated by additional cardiac transcription factors was examined. As expected, in tinman null mutants no cardiac nmr expression is observed, since the cardiogenic region is not specified. Mesodermal overexpression of tinman, however, is unable to induce ectopic nmr expression, suggesting other factors may cooperate together with tinman to initiate nmr expression. Indeed, when tinman was overexpressed together with pannier throughout the mesoderm, significantly more ectopic nmr expression was observed than with pannier alone, suggesting that tinman and pannier act synergistically to induce nmr and other cardiac marker gene expression. Dmef2 mutants were also examined. In Dmef2 mutants muscle myosin fails to be expressed. nmr was found to be expressed normally, consistent with Dmef2's role in myocardial differentiation rather than in specification of cardiac progenitors (Qian, 2005).
In summary, nmr1−,nmr2meso− embryos, one subpopulation of cardiac progenitors, the Eve cluster, is expanded within the cardiogenic region while another set of cardiac progenitors, the Lbe cluster, is reduced. In addition, nmr genetically interacts with pannier by synergistically aggravating pannier's cardiac phenotype. These findings in conjunction with the timing and pattern of expression suggest that nmr participates, along with tinman and pannier, in the initial specification and cell type allocation within the heart-forming mesoderm (Qian, 2005).
The switch in cardiac cell type specification due to loss-of-nmr-function is reminiscent of the phenotype observed in embryos with increased Hedgehog or Ras signaling, in which lbe expression is eliminated while eve is greatly expanded within the cardiogenic mesoderm. Hedgehog signaling seems to regulate the expression level of the EGF receptor ligand protease, encoded by rhomboid (rho), thus activating Ras signal transduction. This raises the possibility that Nmr may act as a link between ectodermal Hedgehog signaling and cardiac patterning. It would be interesting to find out whether Hedgehog signaling limits the expression or activity of mesodermal nmr, which in turn might antagonize Ras signaling to prevent formation of Eve-expressing progenitors. Interestingly, not only is eve expression abolished upon nmr overexpression, but ectopic Eve can also suppress nmr expression, suggesting that there is a reciprocal antagonism between nmr and eve, as between lbe and eve. It will be interesting to find out if nmr expression is expanded in eve mutants, in which mesodermal eve expression is selectively eliminated (Qian, 2005).
The initially expanded size of the Eve progenitor clusters in nmr1−,nmr2meso− embryos does not persist to the same extent after the germ band has retracted and the Eve paracardial cells and muscle founder cells have begun to differentiate. It is possible that from the postulated pre-muscular Eve cluster, although initially enlarged in nmr mutants, a normal number of progenitors are selected. Alternatively, there may be fewer cell divisions by the Eve progenitors, consistent with a possible role of Tbx20 in proliferation. In contrast, another type of emerging pericardial cells, marked by odd expression, remains expanded. These progenitors emerge at a later time than the Eve cells and are possibly subject to a different mechanism of selection and differentiation (Qian, 2005).
The processes of vertebrate cardiac morphogenesis are rather complex and not very well understood beyond a descriptive level. The Drosophila heart, in contrast, is remarkably simple but nevertheless resembles that of vertebrates in its initial assembly from bilateral primordial tissue into a highly organized linear vessel-like tube. It is thus of interest to understand the control mechanisms by which a primitive heart tube forms. The defects in myocardial alignment, heart tube assembly and proper polarity acquisition observed in nmr mutants indicate that these T-box genes play a crucial role during these processes of cardiac morphogenesis. Moreover, when nmr2RNAi is specifically targeted to the differentiating myocardial cells, the overall alignment of the bilateral myocardial primordia at the dorsal midline is little affected but the correct epithelial polarity is not established. Together, these observations further suggest that cardiac determination and distinction of cell types are controlled by mechanisms that are separable and perhaps independent from those that govern heart tube assembly and cellular polarity prerequisite for morphogenesis. It will be interesting to find out what transcriptional targets nmr controls at progressively later stages of heart development, and whether vertebrate Tbx20 also influences multiple steps during heart development (Qian, 2005).
This report provides the first evidence that nmr/Tbx20 also has a role in regulating myocardial polarization, in addition to its role in cardiac cell specification. Studies of cell polarity in other tissues, such as salivary gland, oocyte, neuroblast, suggest cell polarization is important for organelle transport and cell-cell communication via adherens junctions or extracellular matrix components. Thus, a failure in proper polarity acquisition is likely to contribute to the observed disorganization of the heart tube. Since only some of the polarity markers tested exhibit a typical epithelial subcellular distribution in the forming myocardium, it is speculated that there may be significant differences by which the heart epithelium is involved in this organ's morphogenesis. Further explorations into the process of cardiac cell polarization are necessary to obtain a mechanistic understanding of cardiac morphogenesis (Qian, 2005).
Genetic analysis demonstrates a regulatory interaction between nmr and other pivotal factors involved in Drosophila heart development, namely pannier and tinman. For example, loss-of-pannier-function abolishes mesodermal nmr expression, while pan-mesodermal overexpression of pannier results in ectopic nmr expression, which is synergistically augmented by co-expression of tinman. This is consistent with the idea that nmr is a transcriptional target of the cardiac determinants, Pannier and Tinman. Interestingly, the emerging cardiac cell types exhibit a differential susceptibility to pannier; pericardial Eve being the least sensitive, which is consistent with the absence of GATA consensus binding sites in the mesodermal eve enhancer (Qian, 2005).
nmr does not only appear to be a target of pannier but also interacts with pannier genetically in the overall formation of cardiac mesoderm: nmr1;pnr double mutants have a more dramatic reduction in heart-associated cells stained with Tinman or Eve than pannier single mutants (nmr1 single mutants do not exhibit a cell specification phenotype). This indicates that nmr also participates in the initial specification of cardiac competence, but this particular role of nmr is redundant in the presence of pannier, since nmr1−,nmr2meso− embryos initially have a normal level of cardiac-restricted tinman expression. This idea is supported by the finding that overexpression of nmr is sufficient to drive ectopic tinman expression. Thus, it appears that nmr has two, in part opposing, roles during early cardiac specification: it acts redundantly with pannier to specify tinman-expressing cardiac mesoderm from which all cardiac cell types emerge, but also acts in distinguishing subpopulations of cardiac progenitors (Lbe versus Eve cell fates), perhaps by negatively mediating positional information provided by the ectodermal Hedgehog signal (Qian, 2005).
Tbx20 exhibits prominent expression in the heart of all vertebrate and invertebrate organisms examined, and phylogenetic analysis places Tbx20 within the Tbx1 subfamily that is distinct from Tbx5, suggesting evolutionary conservation. In Zebrafish, Tbx20/hrT is co-expressed with Nkx2-5 and Gata4 in cardiogenic as well as in non-cardiac mesoderm adjacent to the tail bud (Szeto, 2002). Since a genetic interaction is observed in Drosophila between Tbx20/nmr and Nkx2-5/tinman, Gata4/pannier, it may be that a similar interaction takes place in vertebrates. Morpholino knock-down of Tbx20/hrT in zebrafish apparently affects chamber morphology, which is consistent with observations of morphogenesis defects in Drosophila nmr mutants. Whether Tbx20 plays a role in cardiac cell polarization in vertebrates remains to be determined. In chicken, Tbx20 is expressed in a complementary pattern to Tbx5 in the embryonic ventricles, and in ovo electroporation and cell culture studies suggest an antagonistic relationship between them (Plageman, 2004). This is reminiscent of the complementary expression and antagonism observed between nmr and eve in flies, which lack a Tbx5 homologue. Taken together, these studies suggest that Tbx20 genes are likely to play multiple roles during cardiogenesis, ranging from specification, proliferation and morphogenesis to cardiac physiology (Qian, 2005).
The Drosophila ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from one another. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. This study shows that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment (Buescher, 2006).
Previous studies of segment polarity gene function have focused mainly on the posterior part of the segment. These studies have revealed an intricate regulatory network involving the wg, engrailed (en), invected (inv), gooseberry (gsb) and patched (ptc) genes underlying the formation and specification of NBs in the posterior part of the segment. By contrast, the formation and specification of NBs in the anterior-most part of the segment are less well understood. This is a study of the segment polarity genes mid and H15, which are expressed in the neuroectoderm (NE) of the anterior- and posterior-most part of each segment. Mutations affecting mid were first identified in a screen for segmentation defects and additional alleles have been identified in screens for axon guidance defects in the peripheral nervous system (lost in space) (Kolodziej, 1995) and the ventral nerve cord (Seeger, 1993). The mid gene was cloned and shown to encode a T-box transcription factor (CG6634). The mid gene is flanked by its paralog H15 (CG6604). This study shows that mid and H15 are largely co-expressed in the NE, several identified NBs and a subset of the putative neuronal and glial progeny of these NBs. mid, but not H15, is required in the NE for the formation and specification of NBs in the anterior-most part of the segment. Furthermore, in individual NBs, mid and H15 have partially redundant functions in the regulation of NB marker gene expression. Accordingly, in the mature ventral nerve cord of midline and H15-deficient embryos, a loss of neurons is found and a misspecification of individual neuronal/glial fates (Buescher, 2006).
The mid RNA expression pattern is typical of segment polarity genes. Briefly, from stage 5 onwards, mid is expressed in 14 stripes, with those in presumptive, odd-numbered abdominal segments being wider and more intensely stained than those in even-numbered abdominal segments. Up to stage 9 mid RNA is co-expressed with En, but also extends two to three rows of cells posteriorly to the En domain in the odd-numbered stripes. During stage 9, these odd-numbered stripes narrow, and from late stage 9 until the end of stage 11, mid colocalizes exactly with the En domain in all stripes. During stage 12, mid expression within the En domain decays and is subsequently re-initiated in a narrow stripe posterior to the En domain; this expression is maintained until the end of embryogenesis. Hence, mid RNA is found in the NE from which early and late born (SI-SV) row 6 and row 7 NBs arise and (in odd-numbered abdominal segments) from which early born (SI-SII) row 1/2 NBs derive (the row of NBs posterior to the En domain is referred to as row 1/2, because it produces row 1 and row 2 NBs). The mid gene is flanked by its paralog H15 which has previously been shown to have partially redundant function with mid. Using an H15-specific RNA probe, it was determined that H15 RNA is first observed in segmentally repeated stripes at stage 7: this is considerably later than mid and initially at a much lower level than mid expression although at later stages, H15 expression levels increase and from stage 9 onwards are comparable to mid expression levels. Like early mid stripes (prior to stage 9), early H15 stripes in presumptive odd-numbered abdominal segments are wider and more intensely stained than stripes in even-numbered segments (Buescher, 2006).
To examine mid/H15 expression in NBs, wt embryos were stained with a mid-specific RNA in situ probe and the viable lacZ enhancer trap H15 was stained with anti–β-galactosidase (βgal). mid-specific RNA in situ studies showed that all NBs that delaminate from mid-positive NE transiently express mid but in most NBs expression rapidly decays. However, two SI NBs and one SII NB accumulate and maintain high levels of mid RNA while a second SII NB maintains lower levels of mid expression. These mid-positive NBs appear posteriorly adjacent to the Wg expression domain. H15-lacZ expression in NBs is indistinguishable from that of mid. To identify the mid/H15-positive SI NBs, double label experiments were performed with an antibody against Worniu (Wor), which is expressed in all NBs. At early stage 9, anti-Wor labels nine SI NBs which can be identified based on their positions and it was possible to identify the mid-positive NBs as NB2-5 and NB7-4. To identify the two SII mid/H15-positive NBs, their positions were mapped along the AP and DV axes relative to the En stripe and the NB marker gene eagle (eg). The results indicate that these SII NBs are En-positive and form medially, adjacent to the Eg-positive NB7-3, identifying the mid/H15-positive SII NBs as NB1-2 and NB7-2. From stage 10 onwards, mid/H15 expression was also found at the ventral midline: time of birth, position and the co-expression of En identify this cell/cell cluster as the MNB or its early progeny (Buescher, 2006).
Expression of mid/H15 in the developing nerve cord increases up to stage 14 when each hemisegment contains approximately 50 (thorax) or 40 (abdomen) mid/H15-positive cells. After stage 14, some mid expression decays so that at the end of embryogenesis (stage 16/17) all hemisegments contain approximately 15 cells which express mid. H15 RNA is observed in a similar pattern. By contrast, at stage 16, H15lacZ expression is found in a larger number of cells, probably reflecting the perdurance of LacZ protein (Buescher, 2006).
Most mid/H15-positive cells are neurons as assayed using the neuron-specific anti-Elav antibody. However, co-expression of H15-lacZ and the glial marker Repo was observed in the dorsal and ventral channel glia and two lateral subperineural glia. These cells also express En, suggesting that they are identical to the previously described En-positive glial progeny of NB7-4 (Buescher, 2006).
mid/H15 and En are co-expressed in NBs 1-2, 7-2 and 7-4. However, co-expression in most of their progeny is transient and in the stage 16 lateral nerve cord is restricted to two to three cells, including the lateral subperineural glia. A similar situation was found at the ventral midline: the MNB stains positively for mid RNA/H15lacZ and En protein and at stage 14 the En-positive progeny of the MNB express mid/H15. However, mid/H15 expression subsequently decays and at stage 16 mid/H15 expression at the ventral midline is restricted to the dorsal and ventral channel glia. Notably, mid/H15lacZ expression was never observed in the midline glia which derive from the region anterior to the En domain (Buescher, 2006).
This analysis of the mid/H15 expression patterns demonstrates that these genes are co-expressed in the NE from which row 6, row 7 and early row 1/2 NBs arise. mid/H15 are also co-expressed in the MNB, NB7-2, NB7-4, NB1-2 and NB2-5 and at least a subset of their progeny. Due to the lack of appropriate markers, the lineages of NB7-2, NB1-2 and NB2-5 have remained among the least studied lineages in the VNC. The observation that these NBs are part of a very small group of mid/H15 expressing NBs will greatly facilitate the analysis of their respective lineages (Buescher, 2006).
To investigate the roles of mid/H15 in the development of the VNC, the mutant phenotypes were studied associated with mid and/or H15 loss-of-function alleles. In this study, the alleles midGA174, mid1, mid2 and H15x4, and a combination of the deficiencies GpdhA and x528 were used. midGA174 and mid1 are EMS-induced, putative amorphic alleles, while mid2 is an EMS-induced strong hypomorphic allele. H15x4 is a deletion of the H15 locus and the combination of deficiencies x528 and GpdhA deletes only mid, H15 and one additional predicted gene (CG31647) of unknown function (Buescher, 2006).
During embryonic stages 9-11, when NB formation takes place, mid/H15 RNA is expressed in the NE from which row 6, row 7 and early row 1/2 NBs arise. To determine whether mid/H15 play a role in the formation of these NBs, mid and H15 single mutant and mid/H15-deficient embryos were examined with the anti-Wor antibody which labels all NBs. At early stage 9, Wor expression in wt embryos is found in four NBs which arise from mid/H15-positive NE: NB1-1 and NB2-5 (row 1/2), and NB7-1 and NB7-4 (row 7). Staining of early stage 9 midGA174 mutant embryos revealed a frequent loss of Wor expression in row 1/2 NBs, with a strong bias towards odd-numbered abdominal segments: for example, NB1-1 was lost in 68.7% of the odd-numbered abdominal segments compared to only 5% loss in even-numbered abdominal segments. NB formation was also affected at the NB2-5 position, albeit less severely; for example: in midGA174 mutant embryos, NB2-5 was lost in 32% of the odd-numbered and 7% of the even-numbered abdominal segments. By contrast, formation of early row 7 NBs was found to be unaffected in midGA174 mutant embryos. Staining of stage 9 mutant embryos with an antibody against Ase, an additional marker for all delaminated NBs, confirmed these results. The neuronal marker gene even-skipped (eve) is not expressed in NBs, but in stage 9 wt embryos, Eve protein is present in the first-born progeny of the SI NBs 7-1 and 1-1; thus the presence of Eve-positive GMCs represents a read-out of NB formation within row 7 and row 1/2. Staining of stage 9 midGA174 mutant embryos revealed that GMC7-1a formed normally but GMC1-1a was frequently absent in odd-numbered segments. This supports the observations that (1) mid function is required for SI NB formation in row 1/2 predominantly in odd-numbered segments and (2) that mid is dispensable for SI NB formation in row 7 (Buescher, 2006).
Previous studies have shown that while some segment polarity genes control the formation of early- and late-born NBs within their activity domain (for example gsb), others are only required for late forming NBs. To investigate whether mid plays any role in the formation of later-born NBs (SII-SV), mid mutant embryos were stained with markers which label either all NBs or identify subsets of NBs. In late stage 11 wt embryos, all NBs have delaminated and form a continuous two-dimensional sheet. In midGA174 mutant embryos, a frequent loss of Wor expression, predominantly in odd-numbered abdominal segments, results in a fragmented appearance of the NB sheet. This indicates that early and late-born NBs are lost in mid mutant embryos. To determine if the loss of mid affects the formation of late-born NBs in rows 6 and 7, NBs were labeled with an antibody against En, which is at least transiently present in all row 6 and row 7 NBs and the MNB. Staining of stage 11 midGA174 mutant embryos did not reveal any loss of En-positive NBs in the lateral CNS indicating that the requirement for mid in NB formation is restricted to row 1/2. However, occasionally a loss/reduction of En expression was observed in the position of the MNB, which forms slightly posterior to the En stripe. This loss/reduction occurred almost exclusively in odd-numbered abdominal segments. The conclusion that mid function is not required for NB formation in rows 6 and 7 was corroborated by examination of the NB marker gene eg. In wt embryos, Eg is expressed in NB7-3, NB6-4, NB2-4 (which all derive from mid/H15-positive NE) and in NB3-3. In mid mutant embryos, a loss of Eg expression was occasionally observed in NB2-4 position (17.8% loss) but not in the NB6-4 and NB7-3 positions. As seen for other row 1/2 NBs, loss of NB2-4 occurred predominantly in odd-numbered abdominal segments. Finally, the formation was monitored of the anterior glioblast which arises in the lateral-most position of row1/2. Staining of stage 10 mid mutant embryos with the glial marker anti-Repo revealed that the glioblast forms normally (Buescher, 2006).
The loss-of-function phenotype of mid with respect to NB formation is only partially penetrant in midGA174 embryos. The most severely affected progenitor cell, NB1-1 in odd-numbered abdominal segments, fails to form in 68.7% of the hemisegments. In 31.3% of the odd-numbered hemisegments, an NB still forms and expresses Wor and Ase. Consistent with the notion that midGA174 and mid1 are amorphic alleles, no significant increase in the loss of NB1-1 was found in midGA174/deficiency or mid1/deficiency hemizygotes. This raises the question of whether the mid paralog H15 can compensate for the requirement for mid. Deletion of the H15 gene alone (H15x4) had no effect on the NB formation. Moreover, removal of both copies of the mid and H15 genes did not appreciably enhance the loss of NBs indicating that H15 plays no role in NB formation even in the absence of mid (Buescher, 2006).
In summary, the results show that mid, but not H15, plays an essential role in the formation of early- and late-born row 1/2 NBs predominantly, but not exclusively, in odd-numbered abdominal segments. This requirement for mid reflects the neuroectodermal expression of mid posterior to the En domain and its segmental differences. Earlier studies had indicated that an additional segment polarity gene, patched (ptc), is required for the formation of row 1/2 NBs. This raises the question of whether mutual regulatory interactions between mid and ptc underlie the requirement for both genes. Loss of mid does not result in changes of the ptc expression pattern, and mid expression is not affected by ptc mutants. Mutation of either mid or ptc alone does not result in a complete loss of NB formation in row 1/2, suggesting that these genes may have partially redundant functions in NB formation. However, in mid/ptc double mutants, odd-numbered segments are shorter than in wild-type embryos by approximately two rows of cells in the neuroectoderm posterior to the en domain (the region from which row 1/2 NBs delaminate). Thus, mid and ptc appear to have redundant functions in maintaining the integrity of the neuroectoderm. This early defect makes a proper interpretation of subsequent events in NB formation impossible (Buescher, 2006).
The defects in NB formation in mid mutant embryos do not reflect the neuroectodermal expression of mid within the En domain where mid function is dispensable. This apparent lack of mid function may be explained by a functional redundancy of individual segment polarity genes with respect to NB formation. A functional redundancy of the hh and wg genes in the formation of NB7-3 has been reported and it is conceivable that segment polarity genes with expression domains overlapping that of mid, such as en and hh, may compensate for the loss of mid. Although it has long been established that loss of segment polarity gene function results in the loss of NBs, the molecular mechanisms by which segment polarity genes promote NB formation have remained unclear. Further studies are required to determine regulatory interactions between the segment polarity genes and the proneural genes of the AS-C and the NB-promoting gene SoxNeuro (Buescher, 2006).
Individual NB identity is specified in the NE by the overlapping expression of segment polarity and dorso-ventral patterning genes; loss of segment polarity gene function results in misspecification of NBs along the antero-posterior axis. mid functions in the NE to break the symmetry of Hh-signaling and loss of mid results in ectopic neuroectodermal Wg expression posterior to the En/Hh domain. In wt embryos, Wg-positive NE gives rise to the row 5 NBs, all of which are Wg-positive at the time of birth. To examine whether the ectopic neuroectodermal expression of Wg results in ectopic Wg expression in NBs, stage 11 mid mutant embryos were stained with the anti-Wg antibody. Wg-positive NBs were observed in row 1/2 predominantly in odd-numbered segments. Ectopic Wg in NBs was only partially penetrant and highly variable with respect to the DV position (identity) of the affected NBs, most probably reflecting the substantial loss of NBs in this region. Removal of both copies of mid and H15 did not enhance the severity of the phenotype. These data show that mid, but not H15, is required to prevent the formation of Wg expressing NBs in row 1/2 in odd-numbered abdominal segments (Buescher, 2006).
Row 1/2 NBs are distinguished from NBs in adjacent rows by the expression of Mirror (Mirr). Mirr expression is initiated during stage 7 in transverse stripes in the NE. Across the ventral midline, the neuroectodermal expression of Mirr extends further anteriorly by 2-3 rows of cells. All row 1/2 NBs, the MNB and NB6-1 are at least transiently Mirr-positive. Staining of stage 7 midGA174 mutant embryos revealed a strong reduction of neuroectodermal Mirr expression. Loss of Mirr expression is more pronounced in odd-numbered segments. In stage 10 midGA174 mutant embryos, a strong loss of Mirr is observed in the row 1/2 NB layer predominantly in odd-numbered abdominal segments; this loss arises through the additive effects of the loss of Mirr expression in the NE (which occurs prior to NB formation) and the general loss of row 1/2 NBs. The severity of the phenotype was not enhanced in mid/H15-deficient embryos. It is noteworthy that the loss of Mirr function itself (mirrcre2) does not result in defects in NB formation suggesting that mid does not promote NB formation via Mirr. In summary, Mirr expression represents a further aspect of row 1/2 NB identity and mid, but not H15, contributes to the specification of this identity by promoting the expression of Mirr in odd- and in (to a lesser extent) even-numbered abdominal segments. The consequences of the loss of Mirr function in NB specification and lineage elaboration are as yet unknown (Buescher, 2006).
To investigate whether loss of mid/H15 results in additional misspecifications of NBs, midGA174 mutant embryos were stained with the anti-Eg antibody. In wt, mid/H15 and Eg expressing NBs are close neighbors but expression of these genes appears mutually exclusive. In mid mutant embryos, ectopic Eg expression was found in NB7-4 (12%), in both odd- and even-numbered abdominal segments. By contrast, removal of the H15 gene alone did not result in ectopic Eg expression. However, removal of both copies of mid and H15 caused an enhancement of the phenotype (37.8%) indicating that in the absence of mid, H15 contributes to the regulation of Eg expression. These results indicate that both mid and, to a lesser extent, H15, act as negative regulators of Eg expression (Buescher, 2006).
A role for mid in the negative regulation of Eg expression was surprising since in wt embryos, two of the Eg-positive NBs (NB6-4 and NB7-3) derive from mid/H15-positive NE. However, in contrast to NB7-4, NB6-4 and NB7-3 rapidly lose mid/H15 expression during NB formation while mid/H15 expression in NB7-4 is not only maintained but also upregulated. These results suggest that, in NB7-3 and NB6-4, mid and Eg expression occurs consecutively while prolonged expression of mid in NB7-4 prevents Eg expression. To test this hypothesis, the expression of mid was extended in NB6-4 and NB7-3 using enGal4 to drive the expression of mid. Staining of stage 11 embryos with anti-Eg revealed a complete loss of Eg expression in the NB6-4 and NB7-3 positions. In addition, ectopic expression of mid with the pan-neural driver scabrousGal4 (scaGal4;UAS-mid) resulted in a nearly complete absence of Eg expression. These results confirm that high levels of mid negatively regulate the expression of Eg in NBs. The ectopic expression of Eg in mid mutant embryos is restricted to NB7-4, indicating that the absence of mid per se is not sufficient to permit Eg expression. The regulation of Eg expression involves positive acting factors such as Huckebein and En which have locally restricted expression patterns. The finding that mid negatively regulates Eg in NB7-4 indicates that Eg expression in individual NBs is achieved by a combinatorial code of positively and negatively acting factors (Buescher, 2006).
In segment polarity gene loss-of-function mutants, NBs are often misspecified, such that NBs which form in one particular row adopt the identity of NBs which develop in an adjacent row. Earlier studies have suggested that row 3/4 NB specification versus row 1/2 specification is accomplished by Wg signaling, which is thought to be high in row 3/4 and low or absent in row 1/2. Consistent with such a role for spatially restricted Wg signaling, ectopic Wg expression seems sufficient to induce row 1/2 NBs to acquire row 3/4 identities. In mid mutant embryos, ectopic Wg expression in the NE of row 1/2 appears at stage 9, the time from which SII to SV NBs form. This prompted an examination of whether loss of mid function causes row 1/2 NBs to adopt a row 3/4 identity. To score row 3/4 identity, the neuronal marker protein Eve was examined. Eve is expressed in the progeny of two row 3/4 NBs: the RP2 neuron which derives from NB4-2 and the EL neuron cluster, which derives from NB3-3. Staining of stage 16 midGA174 mutant embryos did not show ectopic EL neuron clusters, indicating that there is no duplication of a NB3-3 identity. However, in mid mutant embryos, a gain of Eve expression was observed in one neuron per hemisegment, located at an antero-posterior position similar to that of RP2. This extra Eve-positive cell is located lateral to the wt RP2 cell and was found in 46% of the hemisegments with no preference for odd-numbered segments. Removal of both copies of mid and H15 resulted in a strong enhancement of the phenotype, indicating that in the absence of mid, H15 plays a role in the restriction of Eve. Using an eveGal4 driver to direct tau-lacZ expression, it was established that the extra Eve-positive cell projects its axon towards the ventral midline, in contrast to the wt RP2 motorneuron, which has a lateral axon projection which exits the VNC. Hence, this cell is not an extra RP2 neuron and does not indicate a duplicated NB4-2 lineage. However, its parental NB could not be identified (Buescher, 2006).
No evidence was found for row 1/2 NBs adopting row 3/4 identities in mid mutant embryos although it is conceivable that ectopic Wg expression in mid mutant embryos occurs too late (stage 9) to cause a change of NB identities (Buescher, 2006).
To assess defects in the mature VNC, mid/H15 mutant embryos were examined with the anti-En antibody. At stage 16 of embryonic development, each wt hemisegment contains three clusters of cells with high levels of En expression: a small cluster of 2-3 En-positive cells at the lateral-most border of the VNC, a larger cluster comprising 5-6 cells is located in a medio-lateral position and 5-7 En-positive neurons and the channel glia at the ventral midline. mid/H15 are co-expressed with En at different stages in neurogenesis and hence loss of mid/H15 may affect NB specification, lineage elaboration and neuronal/glial differentiation. Staining of stage 16 midGA174 mutant embryos with the anti-En antibody revealed a severely aberrant En expression pattern. A frequent loss of the lateral-most En-positive cell clusters was observed, while the En positive medio-lateral cluster contained fewer cells (3-4 instead of 5-6) and the cells were spatially disarranged. At the ventral midline, the number of the En-positive neurons was reduced to 4-5 cells in most segments and completely absent in 12% of the segments. The channel glia frequently showed migration defects or were absent. In H15 single mutant embryos (H15x4), no loss of En-positive neurons was found. However, removal of both copies of mid and H15 enhanced the severity of the mid single mutant phenotype. Defects were observed with similar frequencies in odd- and even-numbered segments reflecting the observation that mid/H15 expression in NBs and their progeny shows no segmental differences (Buescher, 2006).
In summary, mid and, to a lesser extent, H15 play an important role in the formation of the En-positive cells of the VNC. At which step in neurogenesis do mid/H15 facilitate the formation of En expressing neurons? Since mid and H15 are not required for the formation of En-positive NBs, they may act in NBs to control lineage elaboration. Alternatively, mid/H15 may act in post-mitotic cells to maintain the expression of En. These possibilities are not mutually exclusive and require further investigation (Buescher, 2006).
This study has shown that mid and H15 are expressed in the NE from which the NBs of the rows 6, 7 and 1/2 arise, and this expression is maintained in a small subset of NBs and some of their putative progeny. Like many other segment polarity genes, mid has a dual function in the NE: it is required for NB formation within its expression domain (the anterior-most part of the segment). In addition, mid contributes to the specification of row 1/2 NBs by regulating aspects of the gene expression profile characteristic for this row. Analysis of H15 single and H15/mid-deficient embryos indicates that H15 plays no role in these processes although it has some functional activity in later aspects of neurogenesis and other developmental processes. However, even in those instances where H15 is functionally active, it plays a minor role which is only revealed in the absence of mid. For example, loss of mid function alone results in some ectopic expression of Eagle while loss of H15 alone does not. However, concomitant loss of both copies of mid and H15 results in an increase of ectopic Eagle. The differential effect on Eg expression is not due to different expression levels of mid and H15 which are approximately equal in NBs and must reflect the different intrinsic properties of both proteins although the molecular basis of these differences in protein activity is unknown. The lack of any function of H15 in the formation and early specification of NBs is most probably due to the combination of low H15 protein activity and the low level of H15 expression prior to stage 9 (Buescher, 2006).
The studies revealed that the function of mid in NB formation and specification is required predominantly in odd-numbered abdominal segments. Thus, it is likely that additional factor(s) exist that play a similar role, predominantly in even-numbered segments. The Drosophila genome was examined for additional T-box proteins with neuroectodermal expression patterns. The Drosophila genome contains eight known genes which encode T-box proteins: omb, byn, org-1, Doc1, Doc2, Doc3, mid and H15. However, besides mid and H15, none of these are neuroectodermally expressed, suggesting that the factor(s) required in even-numbered segments may not be T-box proteins. As increasingly more expression profiles of individual genes become available, suitable candidates should become obvious (Buescher, 2006).
Reference names in red indicate recommended papers.
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date revised: 20 August 2006
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