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Role of svp in Drosophila Pericardial Cell Growth
# % i # Acta Genetica Sinicn, January 2006, 33 (1): 3 2 4 0
ISSN 0379-4172
Role of svp in Drosophila Pericardial Cell Growth 1
YUAN Wu-Zhou"*, ZHANG Yue-Juan13*,TANG Wen-Xian', WANG Jun', LI Yong-Qing , WANG Yue-Qun', ZHU Chuan-Bing', YANG Hong', WU Xiu-Shan',' , Rolf Bodmer2 1. The Center for Heart Development, the Key Laboratory of the Ministry of Education, College of Life Sciences, Hunan Normal Universiv, Changsha 410081, China;
2. The Burnham Institute, La Jolla, California 92037, USA
Abstract: The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-ZacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth. Key words: seven-up (svp);pericardial cells; EPCs; DA1; cell growth
The Drosophila cardiac tube is comprised of two cell types: the cardioblasts (cardiomyocytes), which are the contractile myoendothelial cells, and the pericardial cells, which are numerous loosely arranged non-myogenic cells that flank the cardioblasts. They extend longitudinally from segment T2 to A7. The linear cardiac tube subdivides into an 'aorta' in the anterior region from T3 to A5, and a 'heart' in the posterior region from A5 to A8 ['I . The dorsal vessel arises from the mesoderm. This germ layer is specified at the beginning of embryogenesis. After a series of specification and differentiation, 16 cells within each cluster give rise to 6 cardial and 10 pericardial cells. Genetic marker analyses have demonstrated that this cell type consists of at least eight subtypes'21.Pattern of gene expression in the cardial and pericardial cells is indi-
cated in Fig. 1". ". MC
PC
Fig. 1 Pattern of gene expression within a hemisegment of Drosophila cardiac tube at stage 16 MC: Myocardial cells; PC: Pericardial cells; TMC: Tinman myocardial cells; TLMC: Tinman and lbe myocardial cells; SMC: Svp myocardial cells; TPC: Tinman pericardial cells; TLPC: Tinman and lbe pericardial cells; SOPC: Odd-skipped and svp pericardial cells; OPC: Odd-skipped pericardial cells; TEPC: Tinman and eve pericardial cells. Note that all pericardial cells identified to date express zfh-1 . Anterior is to the left, and the dorsal midline is to the top.
Received: 2004-05-30; Accepted: 2005-07-29 This Work Was Supported by the National Natural Sciences Foundation of China (No.30270644, 3021010392), the Natural Sciences Foundation of Hum Province of China (No.O2JJY4026), the Foundation of the Department of Education in Hunan Province of China (No.02B017). *Contributed equally to this study
0
Corresponding Author. E-mail: xiushanwu2003 @ y&oo.corn.cn
33
YUAN Wu-Zhou er a/.:Role of svp in Drosophila Pericardial Cell Growth
The cardiac tube is composed of reiterated segments, each containing six pairs of cardiomyocytes, with the exception of the most posterior segment that contains only two pairs of cells[51.In each segment,
forms appear to perform different biological tasks. In addition to regulating cellular growth, ras also regulates cell proliferation, differentiation, transformation and apoptosis"61.The rus family of genes encodes a
four of the six pairs of cardiomyocytes express tinman, and the remaining two posterior pairs of car-
family of highly conserved, low-molecular-weight GTP-binding proteins. In Drosophila, ras oncogene
~. in cardiomyodiomyocytes express ~ v p " - ~Besides
at 85D (ras8SD, also known as rusl and hereafter referred to as ras) appears to be the ortholog of H-rus, Ki-ras, and N-ras found in mammals[171.Ras oncogene at 64B (ras64B, also known as rus2) is the Drosophila counterpart of mammalian R-rus. Recent re-
cytes, svp is expressed in two pairs of pericardial cells which also express odd-skipped ", 'I. Svp may indirectly repress tinman in svp cardioblasts through the regulation of downstream genes that affect tinman expression [lo1. Svp has a role in diversifying cardioblast identi-
ties within each segment"O'l'l. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp is segmentally repeatedly expressed in carioblasts 'll'.
Furthermore, in combination with hh and the ax-
ial information furnished by abd-A, svp is required for functional ostiae to differentiate properly, but it does not participate in the process that leads to differential patterning of the aorta and heart. Some of the earliest progenitors that emerge from the cardiogenic region express the pair-rule homeobox gene even-shpped (eve) in small, segmentally reiterated clusters that later differentiate into Eve positive pericardial cells (EPCs) and the dorsal muscle DA1 [12'. Eve has been proposed to act as an identity factor essential for EPCs to acquire and maintain their identity, as it appears to be essential for them to maintain normal gene expression patterns during subsequent differentiation[ 13-151. Previous data obtained with a temperature sensitive allele of eve indicated that eve function is required for EPC differentiation at the time when Eve protein is accumulating in the early clusters [ I 3 ' . Moreover, mesodermal eve function is necessary for normal cardiac physiology as well as for the formation of the D A 1 and DO2 muscles [I31.
search indicated rasl might promote cell growth in the Drosophila wing, in which cells mutant for rus had decreased growth, whereas activation of ras led to an increase in cell size"']. Thus, in addition to promoting a shortening of G1 and a compensatory increase in 6 2 phase"'], ras may function partly through myc to regulate cell growth. Herein we studied the function of svp in pericardial cell patterning. We found that svp was able to repress eve indirectly in eve pericardial precursor cells and seemed to contribute to the spatial restriction of eve expression. We also observed an increase in cell size of EPCs in svp homozygous mutant embryos, suggesting that svp may have a role in cell growth.
1 Materials and Methods 1 . 1 Generation of P-insertionfly stock P{PZ}svp is a svp stock with an insert of the P-element near the svp gene located in 87B4-5, and it contains a lacZ reporter gene inserted near svp. 1.2 Drosophila culturing and embryo collection Stocks are maintained in 25°C with Cornmeal, Sucrose and Yeast Medium as described in Bloomington stock center (http://flystocks.bio.indiana.edu/harvardfood.htm). The embryos were collected according to
Cell growth is a complex process involving
the standard method"" which is described as the fol-
many signaling pathways. One of the most important proteins involved in cell growth is ras. Ras may acti-
lowing. Two days before staining, 2 to 4-day old flies
vate a number of signalling pathways in cells, such as the RasMAPK pathway. Moreover, different ras iso-
agar-fruit juice plates to collect eggs. The embryos
were starved for 14 h and then transferred to were allowed to develop at 25°C to the proper stage.
%df%%
34
1 . 3 Immunohistochemistryand microscope
The antibody staining was performed as described""
and the protocol is as the following.
Dechorionate the collected eggs in bleach for 2 minutes with 50% Bleach Solution. The embryos were fixed with Fix Solution and washed with PBT three times. Samples were blocked with 2% BSAPBT and,
Acta Genetica Sinica Vo1.33 No.1
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2 Results 2. 1 Svp homozygous mutant embryos showed a disrupted expression pattern In order to see if svp is involved in the patterning of heart precursor cells, the expression patterns of the svp gene in the heart region during embryonic development were examined via a seven-up-LacZ reporter gene. As
incubated with anti-Eve at 4°C overnight. The em-
expected, P-Gal expression in the svp-expressing heart
bryos were washed with PBT three times and incu-
progenitors was detected at stage 11, at which time svp
bated with a goat anti-mouse Ig-HRP (Santa Cruz
was expressed in at least two heart progenitor cells in
Biotechnology) at 4°C for 25 hours. Samples were
each hemisegment. In later stages of embryonic devel-
washed with PBT for three times, 30 minutes each.
opment, the P-Gal expression from svp-LacZ was seen
The embryos were then treated with DAB (Biotech)
in two adjacent pairs of the six bilaterally symmetrical
according the kit protocol and subsequently DehyThe following primary antibodies were used:
pairs of cardioblasts in seven posterior parasegments, plus another single pair situated caudally to these. Furthermore, the expression in the two pairs of pericardial
anti-Eve 1:5 000'211, anti-P-galactosidase (Cappel
cells in each parasegment could also be detected after
Labs) 1:4 000 and anti-DMM (Drosophilu muscle
stage 11, although the expression was weaker in contrast
myosin) 1:1 O O O [ ~ ~ ] .
with cardioblasts (Fig.2, A and C). Fig. 2A-F showed
brated and mounted.
Fig. 2 The expression of the seven-up-LacZ reporter gene in the heart region and its progenitors during embryonic development (Staining with anti-0-Gal) The expression of svp was detected at stage 1 1 . In later stages, 0-Gal expression from svp-LacZ was seen in two adjacent pairs of cardioblasts in seven posterior parasegments and a single pair situated caudally to these (A, C, E, F). Note a weaker expression of svp-LacZ in pericardial cells at these stages. Open arrows mark several examples of cardiaoblasts expressing svp-LacZ. Asterisk indicates a weaker expression of svp-LacZ in pericardial cells. Arrows show the location of the region where muscles are missing. A-D, lateral view of the embryos. Anterior is to the left, and the dorsal midline is to the top. E-F, dorsal view of a stage 13 (E) and a stage 15 (F) embryo, respectively. Scale bars correspond to 50 pm.
35
YUAN Wu-Zhou et ul.:Role of svp in Drosophilu Pericardial Cell Growth
that the cardioblasts pattern of svp-LacZ expression was normal in svp mutant embryos, indicating that the regulatory mechanisms maintaining svp expression in the svy cardioblasts did not require svp activity. However,
mutant phenotypes in pericardial and somatic muscle regions in svp homozygous mutant embryos were observed, suggesting a possible role of svp in the formation of pericardial and muscle cells. 2. 2 The fine patterning of EPCs and DA1 cells in svp homozygous mutant embryos Svp-LacZ homozygous embryos exhibited mutant phenotypes in EPCs and DAl cells region. In order to explore the role of svp in detail during EPCs patterning, a loss-of -fuction assay of svp was performed using
P-element insertion, and the EPC expression patterns in svp mutant embryos were examined. At stage 11, mesodermal eve expression began in segmentally repeated clusters of cells (Fig. 3G). Each of these clusters gave rise to two EPCs and a founder of the DA1 muscle (Fig. 3: A, C, E, G and H). In svp homozygous mutant embryos, the formation of progenitors was disrupted in some segments at early stages (Fig. 3B). The EPCs exhibited a disarranged expression pattern (Fig. 3, D and F). Furthermore, the DAl muscle founders were absent in some segments (Fig. 3, D and F). Since svp homozygous mutant embryos display abnormality in EPCs and DAl at early stages, it seems possible that svp might function in the earliest
Fig. 3 In situ hybridization of eve in svp mutant embryos (Stained with anti-Eve) At stage 11, mesoderm;?! eve-positive cells were present in segmentally repeated clusters of cells (G). A and B were embryos at stage 12, C and D were at stage 13, E and F were at stage 14, and H was at stage 16. There was normal expression of Eve in two EPCs and the DAI muscle founders in each segment at diverse stages (A, C, E, G, H). Svp homozygous mutant embryos displayed disarranged expression pattern in EPCs, and partly absent DAl muscles (B, D, F). Arrows indicate the missing DA1 muscle founders (D, F). Open arrows suggest the variation of EPC numbers between the svp wild-type and the homozygous mutant embryos. Note the abnormal expression patterns of eve-positive cells occurring from the early stage 11 to subsequent stages. Anterior is to the left, and the dorsal midline is to the top. Scale bars correspond to 50 pm,
Bj$%$
36
progenitors that emerge from the cardiogenic region at early stages, and it influences the expression pattern of EPCs and the formation of DA1 in later stages. However data from a previous study[*] suggest that svp does not contribute to EPCs/DAl differentiation. We propose that the abnormality of EPCs and DA1 muscles might due to interference of another signaling pathway related with svp, which might also influence the EPCs patterning and the formation of DA1 muscles. 2. 3 The fine patterning of dorsal muscles in svp homozygous mutant embryos Svp-LacZ homozygous embryos showed some mutant phenotypes in somatic muscle regions as well. In order to explore the function of svp in the muscle cells in detail, we checked the state of muscle forma-
Acta Genetica Sinica Vo1.33 No.1
2006
tion in homozygous mutant embryos by DMM antibody staining (Fig.4). The muscle disruption seemed to occur randomly within the homozygous mutant embryos (Fig.4B). Although we still do not know if the defect was due to a failure of muscle formation or a failure of muscle attachment, the function of svp is indeed required in the normal development of dorsal muscles. Considering the absence of DA1 muscles in the svp homozygous mutant embryos, we propose that those abnormalities of dorsal muscles and DA1 muscles might due to the disruption of muscle morphogenesis. 2.4
Alteration of cell size in svp mutant embryos
In svp heterozygous embryos, EPCs almost had the same size with each other (FigSA). However, in svp homozygous mutant embryos, except for a disar-
Fig. 4 Anti-DMM staining of dorsal muscles A shows the normal phenotype of somatic muscles. The svp homozygous mutant embryo exhibits an irregular arrangement of dorsal muscles (B), as noted by arrows. Note the difference between A and B. The visceral muscles are normal in the svp homozygous mutant embryos (C, D). Scale bars correspond to 50 pm.
Fig. 5 Cell size alteration in svp homozygous mutant embryos A shows the normal size of EPCs in wild type embryos ,B,C,D are svp homozygous mutant embryos, it clearly shows some EPCs were larger than adjacent cells. Arrows indicat the enlarged EPCs. Scale bars correspond to 20 pm.
31
YUAN Wu-Zhou et al.:Role of svp in Drosophila Pericardial Cell Growth
ranged expression pattern of EPCs, the size of some EPCs was at least twice larger than the others (Fig. 5: B, C and D). Since ras regulates cellular growth, cell proliferation and differentiation, the increase of EPCs size might due to a disturbance of interaction between svp and rus, or due to the disruption of proper cell divisions. Recent data indicated that regulators of pattern
rial code of patterned gene activity for its highly restricted and stereotyped pattern of expression. Wg and dpp signaling from the ectoderm provides activating inputs, determining where within the mesoderm eve expression is initiated, whereas tinman, activated by twist, provides the mesodermal context in which these inputs are interpreted[251.Wg- and dpp-dependent activation of the rus pathway also contributes to the
formation must participate in defining organ size. However, other experimental data suggested pattern-
level of eve activation[261.
ing genes are not the sole players in monitoring organ
additional mechanisms. First, lateral inhibition medi-
size. The mechanism separable from pattern formation most likely exists to regulate organ size. Here we
ated by Notch, along with cross talk between the
The restricted eve pattern is likely due to two
Notch and rus pathways, result in the selection of two
provided evidence that the mutation of svp may have an effect on the size of EPCs, but whether it is in-
Eve progenitors per segmental cluster, which then
volved in the regulation of organ size requires more evidence.
transcription anterior and posterior to the Eve clusters, repressive mechanisms appear to be at work. Our data
divide asymmetrically [26-281. Second, to prevent eve
show that there might exist a svp-related molecular
3 Discussion The Drosophila heart is a highly organized linear tube located beneath the dorsal midline. The cardiac precursors are specified at the dorsal margin of the trunk mesoderm, giving rise to distinct cell types that are arranged in a segmentally repeated pattern. The molecular processes involved in cardiac mesoderm formation have been studied in some detail [2,5,20-241 A . umber of signaling pathways and transcription factors have been shown to function in these processes, leading to models of a hierarchical network of genetic interactions that govern mesoderm differentiation and heart development. Svp is homologous to the vertebrate COUP-TF transcription factor. It has a universal function in various organs, in addition to diversifying cardioblast identities within each segment””. Our study has identified a role of svp in the pattern of pericardial cells and muscle formation. And we also found an unexpected function of svp in EPC cell growth.
3. 1 The influence of svp in the EPCs and dorsal muscles
pathway that could influence EPC patterns since early differentiation of cell clusters at early stages. The amorphic mutations of EPCs and an absence of DA1 muscles in svp homozygous mutants are probably due to the disruption of svp suppression on the rus pathway. We propose that svp influences the wg-dependent activation of the rus pathway through a direct interaction with rus. Without the indirect repression of svp to eve, eve expands its expression, which results in the disarranged expression pattern of EPCs. So the amorphic mutations and the increase in numbers of EPCs might due to the disruption of svp repression in EPC differentiation. Moreover, the disruption of svp repression on the rus pathway might result in an increase in the size of some EPCs in svp mutant embryos. Previous studies have indicated that ras mutants tend to remain active much longer, so transformation may arise from unregulated stimulation of rus signaling, which either stimulates cell growth or inhibits a p o p t ~ s i s [ ~ ~ ] . Ladybird early (lbe), a homeobox gene expressed adjacent to eve, restricts the positive actions
Eve is an early marker for pericardial cell and
of factors downstream of wg, dpp, and rus to generate the eve patternL2]. Maybe there exists some interaction
dorsal muscle progenitors. Eve requires a combinato-
between svp and lbe, which restricts the expression
38
pattern of eve in the cardiac tube. However, this hypothesis needs more evidence. Both Svp and the ETS domainprotein PointedP2 (PntP2) are effectors of the EGF pathway, which functions downstream of EGFWras ing galling'^@^^'. Whether there exists a signaling pathway contributing to the cell growth of EPCs also deserves further study.
3 . 2 Does svp have a role in size control? Svp controls cell proliferation in the developing Malpighian tubules, therefore we hypothesize that it also influences the proliferation or cellular growth in the developing pericardial cells. In homozygous svp mutant embryos, some of the eve-expressing pericardial cells almost doubled their size compared to the others. But whether svp participates in size control is not clear. The effect of cellular growth on organ size is complex: Some changes in cellular growth do not change organ size, whereas other alterations do'331. The ras gene may affect cell There have also been reports that svp interacts genetically with egfr, ras85D (rus oncogene at SSD), rl, sos, csw and 14 other g e n e ~ ' ~ ~ ' . Therefore, the enlarged size of pericardial cells might result from the disruption of the combinatorial action of svp and ras. Recent studies indicated that when ras was overexpressed in clones of the second instar wing disc and examined at the third instar stage, there was an increase in cell size but not an increase in cell numbers, thus the size of the wing was not changed. So alteration in cellular growth in this gene was not sufficient to alter organ size'331.Thus, if svp indeed acts together with rus, it might only affect the size of pericardial cells, but not the size of the cardiac tube. This deduction needs more exploration and more evidence for proof. The increase of EPCs size also mightbe due to the disruption of proper cell divisions in the cells receiving the mitogenic signal, which suggests svp is involved in mediating the mitogenic response. Recently, ras genes have been the subject of intensive research because they are mutated in almost 30% of human cancers'361.In vertebrates, transcrip-
%j%f%
Acta Genetica Sinica Vo1.33 No.1
tion factors COUP-TF
2006
I and COUP-TF I1 are ho-
mologous to the Drosophila svp. There may exist an analogous role for them in regulating cell growth. It is interesting for us to explore the role of svp and ras in cell growth, and to find the possible mechanism between the svp-related rus pathway and cancer.
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Acta Genetica Sinica Vo1.33 No.1
2006
ISSN 0379-4172
Role of svp in Drosophila Pericardial Cell Growth 1
YUAN Wu-Zhou"*, ZHANG Yue-Juan13*,TANG Wen-Xian', WANG Jun', LI Yong-Qing , WANG Yue-Qun', ZHU Chuan-Bing', YANG Hong', WU Xiu-Shan',' , Rolf Bodmer2 1. The Center for Heart Development, the Key Laboratory of the Ministry of Education, College of Life Sciences, Hunan Normal Universiv, Changsha 410081, China;
2. The Burnham Institute, La Jolla, California 92037, USA
Abstract: The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-ZacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth. Key words: seven-up (svp);pericardial cells; EPCs; DA1; cell growth
The Drosophila cardiac tube is comprised of two cell types: the cardioblasts (cardiomyocytes), which are the contractile myoendothelial cells, and the pericardial cells, which are numerous loosely arranged non-myogenic cells that flank the cardioblasts. They extend longitudinally from segment T2 to A7. The linear cardiac tube subdivides into an 'aorta' in the anterior region from T3 to A5, and a 'heart' in the posterior region from A5 to A8 ['I . The dorsal vessel arises from the mesoderm. This germ layer is specified at the beginning of embryogenesis. After a series of specification and differentiation, 16 cells within each cluster give rise to 6 cardial and 10 pericardial cells. Genetic marker analyses have demonstrated that this cell type consists of at least eight subtypes'21.Pattern of gene expression in the cardial and pericardial cells is indi-
cated in Fig. 1". ". MC
PC
Fig. 1 Pattern of gene expression within a hemisegment of Drosophila cardiac tube at stage 16 MC: Myocardial cells; PC: Pericardial cells; TMC: Tinman myocardial cells; TLMC: Tinman and lbe myocardial cells; SMC: Svp myocardial cells; TPC: Tinman pericardial cells; TLPC: Tinman and lbe pericardial cells; SOPC: Odd-skipped and svp pericardial cells; OPC: Odd-skipped pericardial cells; TEPC: Tinman and eve pericardial cells. Note that all pericardial cells identified to date express zfh-1 . Anterior is to the left, and the dorsal midline is to the top.
Received: 2004-05-30; Accepted: 2005-07-29 This Work Was Supported by the National Natural Sciences Foundation of China (No.30270644, 3021010392), the Natural Sciences Foundation of Hum Province of China (No.O2JJY4026), the Foundation of the Department of Education in Hunan Province of China (No.02B017). *Contributed equally to this study
0
Corresponding Author. E-mail: xiushanwu2003 @ y&oo.corn.cn
33
YUAN Wu-Zhou er a/.:Role of svp in Drosophila Pericardial Cell Growth
The cardiac tube is composed of reiterated segments, each containing six pairs of cardiomyocytes, with the exception of the most posterior segment that contains only two pairs of cells[51.In each segment,
forms appear to perform different biological tasks. In addition to regulating cellular growth, ras also regulates cell proliferation, differentiation, transformation and apoptosis"61.The rus family of genes encodes a
four of the six pairs of cardiomyocytes express tinman, and the remaining two posterior pairs of car-
family of highly conserved, low-molecular-weight GTP-binding proteins. In Drosophila, ras oncogene
~. in cardiomyodiomyocytes express ~ v p " - ~Besides
at 85D (ras8SD, also known as rusl and hereafter referred to as ras) appears to be the ortholog of H-rus, Ki-ras, and N-ras found in mammals[171.Ras oncogene at 64B (ras64B, also known as rus2) is the Drosophila counterpart of mammalian R-rus. Recent re-
cytes, svp is expressed in two pairs of pericardial cells which also express odd-skipped ", 'I. Svp may indirectly repress tinman in svp cardioblasts through the regulation of downstream genes that affect tinman expression [lo1. Svp has a role in diversifying cardioblast identi-
ties within each segment"O'l'l. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp is segmentally repeatedly expressed in carioblasts 'll'.
Furthermore, in combination with hh and the ax-
ial information furnished by abd-A, svp is required for functional ostiae to differentiate properly, but it does not participate in the process that leads to differential patterning of the aorta and heart. Some of the earliest progenitors that emerge from the cardiogenic region express the pair-rule homeobox gene even-shpped (eve) in small, segmentally reiterated clusters that later differentiate into Eve positive pericardial cells (EPCs) and the dorsal muscle DA1 [12'. Eve has been proposed to act as an identity factor essential for EPCs to acquire and maintain their identity, as it appears to be essential for them to maintain normal gene expression patterns during subsequent differentiation[ 13-151. Previous data obtained with a temperature sensitive allele of eve indicated that eve function is required for EPC differentiation at the time when Eve protein is accumulating in the early clusters [ I 3 ' . Moreover, mesodermal eve function is necessary for normal cardiac physiology as well as for the formation of the D A 1 and DO2 muscles [I31.
search indicated rasl might promote cell growth in the Drosophila wing, in which cells mutant for rus had decreased growth, whereas activation of ras led to an increase in cell size"']. Thus, in addition to promoting a shortening of G1 and a compensatory increase in 6 2 phase"'], ras may function partly through myc to regulate cell growth. Herein we studied the function of svp in pericardial cell patterning. We found that svp was able to repress eve indirectly in eve pericardial precursor cells and seemed to contribute to the spatial restriction of eve expression. We also observed an increase in cell size of EPCs in svp homozygous mutant embryos, suggesting that svp may have a role in cell growth.
1 Materials and Methods 1 . 1 Generation of P-insertionfly stock P{PZ}svp is a svp stock with an insert of the P-element near the svp gene located in 87B4-5, and it contains a lacZ reporter gene inserted near svp. 1.2 Drosophila culturing and embryo collection Stocks are maintained in 25°C with Cornmeal, Sucrose and Yeast Medium as described in Bloomington stock center (http://flystocks.bio.indiana.edu/harvardfood.htm). The embryos were collected according to
Cell growth is a complex process involving
the standard method"" which is described as the fol-
many signaling pathways. One of the most important proteins involved in cell growth is ras. Ras may acti-
lowing. Two days before staining, 2 to 4-day old flies
vate a number of signalling pathways in cells, such as the RasMAPK pathway. Moreover, different ras iso-
agar-fruit juice plates to collect eggs. The embryos
were starved for 14 h and then transferred to were allowed to develop at 25°C to the proper stage.
%df%%
34
1 . 3 Immunohistochemistryand microscope
The antibody staining was performed as described""
and the protocol is as the following.
Dechorionate the collected eggs in bleach for 2 minutes with 50% Bleach Solution. The embryos were fixed with Fix Solution and washed with PBT three times. Samples were blocked with 2% BSAPBT and,
Acta Genetica Sinica Vo1.33 No.1
2006
2 Results 2. 1 Svp homozygous mutant embryos showed a disrupted expression pattern In order to see if svp is involved in the patterning of heart precursor cells, the expression patterns of the svp gene in the heart region during embryonic development were examined via a seven-up-LacZ reporter gene. As
incubated with anti-Eve at 4°C overnight. The em-
expected, P-Gal expression in the svp-expressing heart
bryos were washed with PBT three times and incu-
progenitors was detected at stage 11, at which time svp
bated with a goat anti-mouse Ig-HRP (Santa Cruz
was expressed in at least two heart progenitor cells in
Biotechnology) at 4°C for 25 hours. Samples were
each hemisegment. In later stages of embryonic devel-
washed with PBT for three times, 30 minutes each.
opment, the P-Gal expression from svp-LacZ was seen
The embryos were then treated with DAB (Biotech)
in two adjacent pairs of the six bilaterally symmetrical
according the kit protocol and subsequently DehyThe following primary antibodies were used:
pairs of cardioblasts in seven posterior parasegments, plus another single pair situated caudally to these. Furthermore, the expression in the two pairs of pericardial
anti-Eve 1:5 000'211, anti-P-galactosidase (Cappel
cells in each parasegment could also be detected after
Labs) 1:4 000 and anti-DMM (Drosophilu muscle
stage 11, although the expression was weaker in contrast
myosin) 1:1 O O O [ ~ ~ ] .
with cardioblasts (Fig.2, A and C). Fig. 2A-F showed
brated and mounted.
Fig. 2 The expression of the seven-up-LacZ reporter gene in the heart region and its progenitors during embryonic development (Staining with anti-0-Gal) The expression of svp was detected at stage 1 1 . In later stages, 0-Gal expression from svp-LacZ was seen in two adjacent pairs of cardioblasts in seven posterior parasegments and a single pair situated caudally to these (A, C, E, F). Note a weaker expression of svp-LacZ in pericardial cells at these stages. Open arrows mark several examples of cardiaoblasts expressing svp-LacZ. Asterisk indicates a weaker expression of svp-LacZ in pericardial cells. Arrows show the location of the region where muscles are missing. A-D, lateral view of the embryos. Anterior is to the left, and the dorsal midline is to the top. E-F, dorsal view of a stage 13 (E) and a stage 15 (F) embryo, respectively. Scale bars correspond to 50 pm.
35
YUAN Wu-Zhou et ul.:Role of svp in Drosophilu Pericardial Cell Growth
that the cardioblasts pattern of svp-LacZ expression was normal in svp mutant embryos, indicating that the regulatory mechanisms maintaining svp expression in the svy cardioblasts did not require svp activity. However,
mutant phenotypes in pericardial and somatic muscle regions in svp homozygous mutant embryos were observed, suggesting a possible role of svp in the formation of pericardial and muscle cells. 2. 2 The fine patterning of EPCs and DA1 cells in svp homozygous mutant embryos Svp-LacZ homozygous embryos exhibited mutant phenotypes in EPCs and DAl cells region. In order to explore the role of svp in detail during EPCs patterning, a loss-of -fuction assay of svp was performed using
P-element insertion, and the EPC expression patterns in svp mutant embryos were examined. At stage 11, mesodermal eve expression began in segmentally repeated clusters of cells (Fig. 3G). Each of these clusters gave rise to two EPCs and a founder of the DA1 muscle (Fig. 3: A, C, E, G and H). In svp homozygous mutant embryos, the formation of progenitors was disrupted in some segments at early stages (Fig. 3B). The EPCs exhibited a disarranged expression pattern (Fig. 3, D and F). Furthermore, the DAl muscle founders were absent in some segments (Fig. 3, D and F). Since svp homozygous mutant embryos display abnormality in EPCs and DAl at early stages, it seems possible that svp might function in the earliest
Fig. 3 In situ hybridization of eve in svp mutant embryos (Stained with anti-Eve) At stage 11, mesoderm;?! eve-positive cells were present in segmentally repeated clusters of cells (G). A and B were embryos at stage 12, C and D were at stage 13, E and F were at stage 14, and H was at stage 16. There was normal expression of Eve in two EPCs and the DAI muscle founders in each segment at diverse stages (A, C, E, G, H). Svp homozygous mutant embryos displayed disarranged expression pattern in EPCs, and partly absent DAl muscles (B, D, F). Arrows indicate the missing DA1 muscle founders (D, F). Open arrows suggest the variation of EPC numbers between the svp wild-type and the homozygous mutant embryos. Note the abnormal expression patterns of eve-positive cells occurring from the early stage 11 to subsequent stages. Anterior is to the left, and the dorsal midline is to the top. Scale bars correspond to 50 pm,
Bj$%$
36
progenitors that emerge from the cardiogenic region at early stages, and it influences the expression pattern of EPCs and the formation of DA1 in later stages. However data from a previous study[*] suggest that svp does not contribute to EPCs/DAl differentiation. We propose that the abnormality of EPCs and DA1 muscles might due to interference of another signaling pathway related with svp, which might also influence the EPCs patterning and the formation of DA1 muscles. 2. 3 The fine patterning of dorsal muscles in svp homozygous mutant embryos Svp-LacZ homozygous embryos showed some mutant phenotypes in somatic muscle regions as well. In order to explore the function of svp in the muscle cells in detail, we checked the state of muscle forma-
Acta Genetica Sinica Vo1.33 No.1
2006
tion in homozygous mutant embryos by DMM antibody staining (Fig.4). The muscle disruption seemed to occur randomly within the homozygous mutant embryos (Fig.4B). Although we still do not know if the defect was due to a failure of muscle formation or a failure of muscle attachment, the function of svp is indeed required in the normal development of dorsal muscles. Considering the absence of DA1 muscles in the svp homozygous mutant embryos, we propose that those abnormalities of dorsal muscles and DA1 muscles might due to the disruption of muscle morphogenesis. 2.4
Alteration of cell size in svp mutant embryos
In svp heterozygous embryos, EPCs almost had the same size with each other (FigSA). However, in svp homozygous mutant embryos, except for a disar-
Fig. 4 Anti-DMM staining of dorsal muscles A shows the normal phenotype of somatic muscles. The svp homozygous mutant embryo exhibits an irregular arrangement of dorsal muscles (B), as noted by arrows. Note the difference between A and B. The visceral muscles are normal in the svp homozygous mutant embryos (C, D). Scale bars correspond to 50 pm.
Fig. 5 Cell size alteration in svp homozygous mutant embryos A shows the normal size of EPCs in wild type embryos ,B,C,D are svp homozygous mutant embryos, it clearly shows some EPCs were larger than adjacent cells. Arrows indicat the enlarged EPCs. Scale bars correspond to 20 pm.
31
YUAN Wu-Zhou et al.:Role of svp in Drosophila Pericardial Cell Growth
ranged expression pattern of EPCs, the size of some EPCs was at least twice larger than the others (Fig. 5: B, C and D). Since ras regulates cellular growth, cell proliferation and differentiation, the increase of EPCs size might due to a disturbance of interaction between svp and rus, or due to the disruption of proper cell divisions. Recent data indicated that regulators of pattern
rial code of patterned gene activity for its highly restricted and stereotyped pattern of expression. Wg and dpp signaling from the ectoderm provides activating inputs, determining where within the mesoderm eve expression is initiated, whereas tinman, activated by twist, provides the mesodermal context in which these inputs are interpreted[251.Wg- and dpp-dependent activation of the rus pathway also contributes to the
formation must participate in defining organ size. However, other experimental data suggested pattern-
level of eve activation[261.
ing genes are not the sole players in monitoring organ
additional mechanisms. First, lateral inhibition medi-
size. The mechanism separable from pattern formation most likely exists to regulate organ size. Here we
ated by Notch, along with cross talk between the
The restricted eve pattern is likely due to two
Notch and rus pathways, result in the selection of two
provided evidence that the mutation of svp may have an effect on the size of EPCs, but whether it is in-
Eve progenitors per segmental cluster, which then
volved in the regulation of organ size requires more evidence.
transcription anterior and posterior to the Eve clusters, repressive mechanisms appear to be at work. Our data
divide asymmetrically [26-281. Second, to prevent eve
show that there might exist a svp-related molecular
3 Discussion The Drosophila heart is a highly organized linear tube located beneath the dorsal midline. The cardiac precursors are specified at the dorsal margin of the trunk mesoderm, giving rise to distinct cell types that are arranged in a segmentally repeated pattern. The molecular processes involved in cardiac mesoderm formation have been studied in some detail [2,5,20-241 A . umber of signaling pathways and transcription factors have been shown to function in these processes, leading to models of a hierarchical network of genetic interactions that govern mesoderm differentiation and heart development. Svp is homologous to the vertebrate COUP-TF transcription factor. It has a universal function in various organs, in addition to diversifying cardioblast identities within each segment””. Our study has identified a role of svp in the pattern of pericardial cells and muscle formation. And we also found an unexpected function of svp in EPC cell growth.
3. 1 The influence of svp in the EPCs and dorsal muscles
pathway that could influence EPC patterns since early differentiation of cell clusters at early stages. The amorphic mutations of EPCs and an absence of DA1 muscles in svp homozygous mutants are probably due to the disruption of svp suppression on the rus pathway. We propose that svp influences the wg-dependent activation of the rus pathway through a direct interaction with rus. Without the indirect repression of svp to eve, eve expands its expression, which results in the disarranged expression pattern of EPCs. So the amorphic mutations and the increase in numbers of EPCs might due to the disruption of svp repression in EPC differentiation. Moreover, the disruption of svp repression on the rus pathway might result in an increase in the size of some EPCs in svp mutant embryos. Previous studies have indicated that ras mutants tend to remain active much longer, so transformation may arise from unregulated stimulation of rus signaling, which either stimulates cell growth or inhibits a p o p t ~ s i s [ ~ ~ ] . Ladybird early (lbe), a homeobox gene expressed adjacent to eve, restricts the positive actions
Eve is an early marker for pericardial cell and
of factors downstream of wg, dpp, and rus to generate the eve patternL2]. Maybe there exists some interaction
dorsal muscle progenitors. Eve requires a combinato-
between svp and lbe, which restricts the expression
38
pattern of eve in the cardiac tube. However, this hypothesis needs more evidence. Both Svp and the ETS domainprotein PointedP2 (PntP2) are effectors of the EGF pathway, which functions downstream of EGFWras ing galling'^@^^'. Whether there exists a signaling pathway contributing to the cell growth of EPCs also deserves further study.
3 . 2 Does svp have a role in size control? Svp controls cell proliferation in the developing Malpighian tubules, therefore we hypothesize that it also influences the proliferation or cellular growth in the developing pericardial cells. In homozygous svp mutant embryos, some of the eve-expressing pericardial cells almost doubled their size compared to the others. But whether svp participates in size control is not clear. The effect of cellular growth on organ size is complex: Some changes in cellular growth do not change organ size, whereas other alterations do'331. The ras gene may affect cell There have also been reports that svp interacts genetically with egfr, ras85D (rus oncogene at SSD), rl, sos, csw and 14 other g e n e ~ ' ~ ~ ' . Therefore, the enlarged size of pericardial cells might result from the disruption of the combinatorial action of svp and ras. Recent studies indicated that when ras was overexpressed in clones of the second instar wing disc and examined at the third instar stage, there was an increase in cell size but not an increase in cell numbers, thus the size of the wing was not changed. So alteration in cellular growth in this gene was not sufficient to alter organ size'331.Thus, if svp indeed acts together with rus, it might only affect the size of pericardial cells, but not the size of the cardiac tube. This deduction needs more exploration and more evidence for proof. The increase of EPCs size also mightbe due to the disruption of proper cell divisions in the cells receiving the mitogenic signal, which suggests svp is involved in mediating the mitogenic response. Recently, ras genes have been the subject of intensive research because they are mutated in almost 30% of human cancers'361.In vertebrates, transcrip-
%j%f%
Acta Genetica Sinica Vo1.33 No.1
tion factors COUP-TF
2006
I and COUP-TF I1 are ho-
mologous to the Drosophila svp. There may exist an analogous role for them in regulating cell growth. It is interesting for us to explore the role of svp and ras in cell growth, and to find the possible mechanism between the svp-related rus pathway and cancer.
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Acta Genetica Sinica Vo1.33 No.1
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