BACKGROUND
the runt domain  is a highly conserved  <dig> amino acid sequence that defines a metazoan family of sequence-specific dna binding proteins important for the developmental control of cell fate  <cit> .
vertebrates have three runx genes that are essential for the development of specific tissues , each of which is associated with leukemogenic or carcinogenic mutations  <cit> .
some of these mutations cause loss of runx function while others are gain-of-function; hence, mammalian runx genes display attributes of both tumor suppressors and proto-oncogenes  <cit> .
runx proteins have been shown to regulate cell cycle transit via their function as context-dependent transcriptional regulators.
for example, runx <dig> stimulates the g <dig> to s phase transition in cultured mammalian cells, which may in part reflect its transcriptional activation of cyclin d <dig>  <cit> .
runx <dig> is also an transactivator of the p14arf tumor suppressor  <cit> , while both runx <dig> and runx <dig> are transcriptional repressors of the p21cip1/waf <dig> cyclin-dependent kinase inhibitor  <cit> .
these findings indicate that runx is a critical node in the gene regulatory network that controls cell proliferation and/or survival during animal development.
since many of the core linkages in this network are likely to be conserved throughout animal evolution, identification and cis-regulatory analysis of relevant runx target genes in experimentally tractable animal models should help illuminate how runx genes contribute to human development and health.
the sea urchin embryo is optically accessible and highly amenable to experimental manipulations such as gene transfer and morpholino antisense mediated knockdown, making it a useful system for discovering fundamental roles played by runx proteins in the cell biology of animal development.
whole genome sequence data  <cit>  as well as genomic southern blot analysis  <cit>  suggest that, contrary to previous reports  <cit> , the sea urchin strongylocentrotus purpuratus has two runx genes .
only one of these  is expressed in the embryo  <cit> .
sprunt- <dig> is required for embryogenesis beyond blastula stage  <cit>  and contributes to transcriptional activation of the cyiiia actin gene, a marker of aboral ectoderm differentiation  <cit> .
sprunt- <dig> mrna is expressed globally in the early embryo, whereas in the larva it accumulates predominantly in oral ectoderm and endomesoderm, tissues associated with continued growth and cell proliferation  <cit> .
embryos depleted of sprunt- <dig> by morpholino antisense oligonucleotides  that sequence-specifically block either translation or pre-mrna splicing fail to gastrulate normally, display global defects in cell differentiation and proliferation and underexpress a number of genes including that encoding the conventional protein kinase c sppkc <dig>  <cit> .
here we provide evidence that the gastrulation defect and abnormal proliferation associated with sprunt- <dig> deficiency are secondary effects of extensive apoptosis that occurs in the post-blastula stage embryo.
we further demonstrate that apoptosis is caused by a deficit in sppkc <dig> activity, and that cell survival in sprunt- <dig> morphant embryos can be rescued by exogenous sppkc <dig> mrna.
finally, we show that the sppkc <dig> promoter region contains sprunt- <dig> target sequences that are required for its transcriptional activation.
RESULTS
sprunt- <dig> deficiency leads to apoptosis and secondary proliferation
to characterize further the abnormal cell proliferation associated with sprunt- <dig> deficiency  <cit> , gastrula stage embryos injected with either a nonspecific control maso or the splice-blocking sprunt- <dig> maso were pulse-labelled with bromodeoxyuridine .
the embryos were fixed, and an anti-brdu antibody was used to detect brdu incorporation, which is indicative of dna synthesis during the labelling period.
normal late gastrula stage embryos  contained very few cells in s phase, as shown by an absence of nuclear brdu signal in the controls .
in contrast, sprunt- <dig> morphant embryos of the same age  displayed extensive nuclear brdu incorporation throughout the embryo , indicating an abnormally high number of proliferating cells, in agreement with our previous cytometric dna analysis of cell cycle distribution in these embryos  <cit> .
paradoxically, sprunt- <dig> morphant embryos had about half as much dna per embryo as gastrula stage controls of the same age  <cit> .
since deregulated cell proliferation is often associated with programmed cell death  <cit> , we asked whether cells in sprunt- <dig> morphants undergo apoptosis, which could account for these embryos displaying lower than normal dna content despite having ectopic cell cycles.
terminal transferase-mediated dutp nick end labeling  showed that, in contrast to controls , sprunt- <dig> morphants displayed extensive dna fragmentation , a hallmark of apoptosis.
apoptosis was further indicated by the observation that sprunt- <dig> morphants contained elevated levels of activated caspase- <dig>  and caspase- <dig> .
the apoptosis was specifically caused by sprunt- <dig> deficiency, as cell survival is rescued by coinjection of full-length sprunt- <dig> mrna with the splice-blocking sprunt- <dig> maso .
interestingly, rnai-mediated knockdown of the drosophila runx gene cg <dig> was recently shown to lead to the apoptosis of in vitro cultured cells  <cit> ; combined with our data this suggests an evolutionarily fundamental role for runx genes in the control of metazoan cell survival.
ectopic cell cycles in the drosophila eye disc lead to compensatory apoptosis via jun kinase  signaling  <cit> .
we hypothesized that apoptosis in sprunt- <dig> morphant embryos might similarly be a secondary response to unregulated cell proliferation, in which case inhibiting apoptosis might allow unbridled cell proliferation leading to a tumor-like overgrowth phenotype.
to test this we used the caspase- <dig> inhibitor acetyl-asp-glu-val-asp-cho .
surprisingly, this drug rescued gastrulation  in about half of the sprunt- <dig> morphants, allowing development of more normal looking embryos with fully invaginated guts .
the rescued embryos displayed negligible apoptosis when assayed by tunel , confirming the drug's efficacy in inhibiting caspase activity and suggesting that apoptosis is a principal cause of the gastrulation defect associated with sprunt- <dig> deficiency.
apoptosis is also likely to be the underlying cause of the ectopic proliferation observed in these embryos , as sprunt- <dig> morphants rescued with the caspase- <dig> inhibitor displayed negligible brdu incorporation at late gastrula stage, whereas sprunt- <dig> morphants treated with the dna polymerase inhibitor aphidicolin  were not rescued from apoptosis .
it is likely that the ectopic cell cycles associated with sprunt- <dig> deficiency are a regulative response to a loss of embryo integrity, as occurs when cells from dissociated gastrula stage embryos re-enter the cell cycle  <cit> .
apoptosis associated with sprunt- <dig> deficiency is caused by loss of pkc expression
protein kinase c  is a key component of numerous developmental signalling pathways, and the conventional pkc sppkc <dig> was previously shown to be one of a number of genes that are significantly underexpressed in sprunt- <dig> morphant embryos  <cit> .
moreover, mammalian pkcα promotes cell survival  <cit> .
to ask whether the apoptosis associated with sprunt- <dig> deficiency might be caused by the loss of pkc expression, we made use of chelerythrine, a compound that selectively and completely inhibits mammalian pkc activity at a concentration of ~ <dig> μm  <cit> .
embryos treated with 2– <dig> μm chelerythrine from the mesenchyme blastula stage on  displayed gastrulation defects  as well as extensive apoptosis as indicated by tunel  and elevated levels of activated caspase- <dig> .
a similar gastrulation-defective phenotype  and cell survival defect  was obtained by injecting embryos with a maso that targeted the translation initiation codon of sppkc <dig> mrna.
in both cases cell survival  was rescued by treatment with the caspase- <dig> inhibitor  despite the continued presence of active caspase- <dig> .
these data strongly suggest that the deficit in sppkc <dig> expression in sprunt- <dig> morphants might be the underlying cause of their apoptotic phenotype, in which case restoring sppkc <dig> mrna in these embryos should rescue cell survival.
to test this, we coinjected zygotes with the sprunt- <dig> maso along with synthetic capped mrna encoding sppkc <dig>  unlike embryos injected with the sprunt- <dig> maso alone, most of the morphants coinjected with solutions containing 400– <dig> ng/μl of sppkc <dig> mrna gastrulated  and displayed negligible tunel signal , indicating that underexpression of pkc is indeed the principal underlying cause of the apoptosis and resulting gastrulation defects associated with sprunt- <dig> deficiency.
sprunt- <dig> target sequences in the sppkc <dig> promoter region are required for transcriptional activation
our findings raised the question of whether the requirement for sprunt- <dig> in support of pkc expression is direct or indirect.
examination of the 5' flanking sequence of the sppkc <dig> gene revealed the presence of two sequences that match the runx target consensus within  <dig>  kb of the first exon .
fluorescently-labeled oligonucleotide probes representing each of these sites bound sequence-specifically to recombinant sprunt- <dig> in an electrophoretic mobility shift assay .
furthermore, sequences from the sppkc <dig> promoter region were specifically recovered by chromatin immunoprecipitation  using an anti-sprunt- <dig> antibody  <cit> , indicating that sprunt- <dig> is bound in vivo to these sequences at gastrula stage .
to test the functionality of the runx target sequences in this region we constructed a luciferase reporter gene linked to ~ <dig>  kb 5' flanking sequence  of sppkc <dig> .
when introduced into embryos, this sequence activated transcription of the reporter by approximately threefold and in a sprunt- <dig> dependent fashion, as reporter expression was reduced to near baseline levels in sprunt- <dig> morphant embryos .
moreover, base substitutions that eliminate either the proximal or the distal runx target sequences in the sppkc <dig> cis-regulatory domain similarly reduced activity of the reporter, as did combined substitutions that eliminated both target sequences , suggesting that the two runx sites function synergistically rather than additively to activate transcription.
the relatively weak activation of the reporter conferred by the  <dig>  kb sppkc <dig> promoter fragment, together with our previous results showing that endogenous sppkc <dig> expression is reduced approximately seven fold in sprunt- <dig> morphant embryos  <cit> , suggests that there may be additional enhancers outside of the  <dig>  kb fragment that collaborate with sprunt- <dig> in activating transcription of the endogenous gene.
testing this possibility would require cis-regulatory analysis using a larger genomic fragment that contains the sppkc <dig> locus.
nevertheless, the data presented here support the proposition that the direct interaction of sprunt- <dig> with its target sequences in the sppkc <dig> cis-regulatory domain is required for normal levels of sppkc <dig> transcription in the post-blastula stage embryo, which in turn is required to maintain pkc activity at levels sufficient to antagonize caspase activity and suppress apoptosis .
CONCLUSIONS
our results support the idea that, in animal development, cells do not survive by default but rather by actively suppressing apoptosis in response to permissive cell signalling  <cit> .
furthermore, we find that deregulated apoptosis can lead to secondary cell proliferation, similar to the compensatory cell proliferation described by others  <cit> .
in contrast to the latter phenomenon however, we do not observe overproliferation caused by apoptotic cell signalling in the caspase- <dig> inhibited embryos.
while the specific role of sppkc <dig> in suppressing apoptosis is not known, there are several plausible pathways based on well-known functions of mammalian pkcα, most notably its role in phosphorylating and thereby activating the anti-apoptotic protein bcl- <dig>   <cit> .
identification of the relevant substrate and pathway in sea urchins awaits further analysis, as does the basis for the secondary proliferation observed in sprunt- <dig> morphants.
in addition to the conventional pkcs , the pkc family also includes non-conventional and atypical pkcs   <cit> .
we do not yet know how many pkc family members are in s. purpuratus, although available expressed sequence tag  and genomic sequence data suggest that sppkc <dig> is the only cpkc gene expressed in the embryo, and possibly the only cpkc gene encoded in the genome .
we note in closing that mammals have three cpkc genes, pkcα, pkcβ, and pkcγ.
pkcβ is actually pro-apoptotic in myeloid cells, and its gene was recently shown to be directly activated by a human runx homolog, runx <dig>  <cit> .
this suggests that runx-mediated control of cpkc gene expression may be a primitive conserved character  in animals, whereas the functional output of this interaction  varies with context, something that would bear on whether a given runx gene is an oncogene or tumor suppressor.
it will therefore be interesting to determine whether the other mammalian cpkc genes are also direct runx regulatory targets, and whether the runx-dependency of cell survival in drosophila  <cit>  is a function of pkc.

 the runt domain is a highly conserved amino acid sequence that defines a metazoan family of sequence-specific dna binding proteins important for the developmental control of cell fate. vertebrates have three runx genes that are essential for the development of specific tissues, each of which is associated with leukemogenic or carcinogenic mutations. some of these mutations cause loss of runx function while others are gain-of-function; hence, mammalian runx genes display attributes of both tumor suppressors and proto-oncogenes. runx proteins have been shown to regulate cell cycle transit via their function as context-dependent transcriptional regulators. for example, runx stimulates the g to s phase transition in cultured mammalian cells, which may in part reflect its transcriptional activation of cyclin d. runx is also an transactivator of the p14arf tumor suppressor, while both runx and runx are transcriptional repressors of the p21cip1/waf cyclin-dependent kinase inhibitor. these findings indicate that runx is a critical node in the gene regulatory network that controls cell proliferation and/or survival during animal development. since many of the core linkages in this network are likely to be conserved throughout animal evolution, identification and cis-regulatory analysis of relevant runx target genes in experimentally tractable animal models should help illuminate how runx genes contribute to human development and health. the sea urchin embryo is optically accessible and highly amenable to experimental manipulations such as gene transfer and morpholino antisense mediated knockdown, making it a useful system for discovering fundamental roles played by runx proteins in the cell biology of animal development. whole genome sequence data as well as genomic southern blot analysis suggest that, contrary to previous reports, the sea urchin strongylocentrotus purpuratus has two runx genes. only one of these is expressed in the embryo. sprunt- is required for embryogenesis beyond blastula stage and contributes to transcriptional activation of the cyiiia actin gene, a marker of aboral ectoderm differentiation. sprunt- mrna is expressed globally in the early embryo, whereas in the larva it accumulates predominantly in oral ectoderm and endomesoderm, tissues associated with continued growth and cell proliferation. embryos depleted of sprunt- by morpholino antisense oligonucleotides that sequence-specifically block either translation or pre-mrna splicing fail to gastrulate normally, display global defects in cell differentiation and proliferation and underexpress a number of genes including that encoding the conventional protein kinase c sppkc. here we provide evidence that the gastrulation defect and abnormal proliferation associated with sprunt- deficiency are secondary effects of extensive apoptosis that occurs in the post-blastula stage embryo. we further demonstrate that apoptosis is caused by a deficit in sppkc activity, and that cell survival in sprunt- morphant embryos can be rescued by exogenous sppkc mrna. finally, we show that the sppkc promoter region contains sprunt- target sequences that are required for its transcriptional activation.  sprunt- deficiency leads to apoptosis and secondary proliferation to characterize further the abnormal cell proliferation associated with sprunt- deficiency, gastrula stage embryos injected with either a nonspecific control maso or the splice-blocking sprunt- maso were pulse-labelled with bromodeoxyuridine. the embryos were fixed, and an anti-brdu antibody was used to detect brdu incorporation, which is indicative of dna synthesis during the labelling period. normal late gastrula stage embryos contained very few cells in s phase, as shown by an absence of nuclear brdu signal in the controls. in contrast, sprunt- morphant embryos of the same age displayed extensive nuclear brdu incorporation throughout the embryo, indicating an abnormally high number of proliferating cells, in agreement with our previous cytometric dna analysis of cell cycle distribution in these embryos. paradoxically, sprunt- morphant embryos had about half as much dna per embryo as gastrula stage controls of the same age. since deregulated cell proliferation is often associated with programmed cell death, we asked whether cells in sprunt- morphants undergo apoptosis, which could account for these embryos displaying lower than normal dna content despite having ectopic cell cycles. terminal transferase-mediated dutp nick end labeling showed that, in contrast to controls, sprunt- morphants displayed extensive dna fragmentation, a hallmark of apoptosis. apoptosis was further indicated by the observation that sprunt- morphants contained elevated levels of activated caspase- and caspase-. the apoptosis was specifically caused by sprunt- deficiency, as cell survival is rescued by coinjection of full-length sprunt- mrna with the splice-blocking sprunt- maso. interestingly, rnai-mediated knockdown of the drosophila runx gene cg was recently shown to lead to the apoptosis of in vitro cultured cells; combined with our data this suggests an evolutionarily fundamental role for runx genes in the control of metazoan cell survival. ectopic cell cycles in the drosophila eye disc lead to compensatory apoptosis via jun kinase signaling. we hypothesized that apoptosis in sprunt- morphant embryos might similarly be a secondary response to unregulated cell proliferation, in which case inhibiting apoptosis might allow unbridled cell proliferation leading to a tumor-like overgrowth phenotype. to test this we used the caspase- inhibitor acetyl-asp-glu-val-asp-cho. surprisingly, this drug rescued gastrulation in about half of the sprunt- morphants, allowing development of more normal looking embryos with fully invaginated guts. the rescued embryos displayed negligible apoptosis when assayed by tunel, confirming the drug's efficacy in inhibiting caspase activity and suggesting that apoptosis is a principal cause of the gastrulation defect associated with sprunt- deficiency. apoptosis is also likely to be the underlying cause of the ectopic proliferation observed in these embryos, as sprunt- morphants rescued with the caspase- inhibitor displayed negligible brdu incorporation at late gastrula stage, whereas sprunt- morphants treated with the dna polymerase inhibitor aphidicolin were not rescued from apoptosis. it is likely that the ectopic cell cycles associated with sprunt- deficiency are a regulative response to a loss of embryo integrity, as occurs when cells from dissociated gastrula stage embryos re-enter the cell cycle. apoptosis associated with sprunt- deficiency is caused by loss of pkc expression protein kinase c is a key component of numerous developmental signalling pathways, and the conventional pkc sppkc was previously shown to be one of a number of genes that are significantly underexpressed in sprunt- morphant embryos. moreover, mammalian pkcα promotes cell survival. to ask whether the apoptosis associated with sprunt- deficiency might be caused by the loss of pkc expression, we made use of chelerythrine, a compound that selectively and completely inhibits mammalian pkc activity at a concentration of ~ μm. embryos treated with 2– μm chelerythrine from the mesenchyme blastula stage on displayed gastrulation defects as well as extensive apoptosis as indicated by tunel and elevated levels of activated caspase-. a similar gastrulation-defective phenotype and cell survival defect was obtained by injecting embryos with a maso that targeted the translation initiation codon of sppkc mrna. in both cases cell survival was rescued by treatment with the caspase- inhibitor despite the continued presence of active caspase-. these data strongly suggest that the deficit in sppkc expression in sprunt- morphants might be the underlying cause of their apoptotic phenotype, in which case restoring sppkc mrna in these embryos should rescue cell survival. to test this, we coinjected zygotes with the sprunt- maso along with synthetic capped mrna encoding sppkc unlike embryos injected with the sprunt- maso alone, most of the morphants coinjected with solutions containing 400– ng/μl of sppkc mrna gastrulated and displayed negligible tunel signal, indicating that underexpression of pkc is indeed the principal underlying cause of the apoptosis and resulting gastrulation defects associated with sprunt- deficiency. sprunt- target sequences in the sppkc promoter region are required for transcriptional activation our findings raised the question of whether the requirement for sprunt- in support of pkc expression is direct or indirect. examination of the 5' flanking sequence of the sppkc gene revealed the presence of two sequences that match the runx target consensus within kb of the first exon. fluorescently-labeled oligonucleotide probes representing each of these sites bound sequence-specifically to recombinant sprunt- in an electrophoretic mobility shift assay. furthermore, sequences from the sppkc promoter region were specifically recovered by chromatin immunoprecipitation using an anti-sprunt- antibody, indicating that sprunt- is bound in vivo to these sequences at gastrula stage. to test the functionality of the runx target sequences in this region we constructed a luciferase reporter gene linked to ~ kb 5' flanking sequence of sppkc. when introduced into embryos, this sequence activated transcription of the reporter by approximately threefold and in a sprunt- dependent fashion, as reporter expression was reduced to near baseline levels in sprunt- morphant embryos. moreover, base substitutions that eliminate either the proximal or the distal runx target sequences in the sppkc cis-regulatory domain similarly reduced activity of the reporter, as did combined substitutions that eliminated both target sequences, suggesting that the two runx sites function synergistically rather than additively to activate transcription. the relatively weak activation of the reporter conferred by the kb sppkc promoter fragment, together with our previous results showing that endogenous sppkc expression is reduced approximately seven fold in sprunt- morphant embryos, suggests that there may be additional enhancers outside of the kb fragment that collaborate with sprunt- in activating transcription of the endogenous gene. testing this possibility would require cis-regulatory analysis using a larger genomic fragment that contains the sppkc locus. nevertheless, the data presented here support the proposition that the direct interaction of sprunt- with its target sequences in the sppkc cis-regulatory domain is required for normal levels of sppkc transcription in the post-blastula stage embryo, which in turn is required to maintain pkc activity at levels sufficient to antagonize caspase activity and suppress apoptosis.  our results support the idea that, in animal development, cells do not survive by default but rather by actively suppressing apoptosis in response to permissive cell signalling. furthermore, we find that deregulated apoptosis can lead to secondary cell proliferation, similar to the compensatory cell proliferation described by others. in contrast to the latter phenomenon however, we do not observe overproliferation caused by apoptotic cell signalling in the caspase- inhibited embryos. while the specific role of sppkc in suppressing apoptosis is not known, there are several plausible pathways based on well-known functions of mammalian pkcα, most notably its role in phosphorylating and thereby activating the anti-apoptotic protein bcl-. identification of the relevant substrate and pathway in sea urchins awaits further analysis, as does the basis for the secondary proliferation observed in sprunt- morphants. in addition to the conventional pkcs, the pkc family also includes non-conventional and atypical pkcs. we do not yet know how many pkc family members are in s. purpuratus, although available expressed sequence tag and genomic sequence data suggest that sppkc is the only cpkc gene expressed in the embryo, and possibly the only cpkc gene encoded in the genome. we note in closing that mammals have three cpkc genes, pkcα, pkcβ, and pkcγ. pkcβ is actually pro-apoptotic in myeloid cells, and its gene was recently shown to be directly activated by a human runx homolog, runx. this suggests that runx-mediated control of cpkc gene expression may be a primitive conserved character in animals, whereas the functional output of this interaction varies with context, something that would bear on whether a given runx gene is an oncogene or tumor suppressor. it will therefore be interesting to determine whether the other mammalian cpkc genes are also direct runx regulatory targets, and whether the runx-dependency of cell survival in drosophila is a function of pkc. 
to discover fundamental runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin runx protein sprunt- previously we showed that embryos depleted of sprunt- arrest development at early gastrula stage and underexpress the conventional protein kinase c sppkc runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. we report here that sprunt- deficiency leads to ectopic cell proliferation and extensive apoptosis. our results show that runx-dependent activation of sppkc is essential for maintaining protein kinase c activity at levels conducive to cell survival during embryogenesis. inhibition or knockdown of sppkc also causes apoptosis, while cell survival is rescued in sprunt- morphant embryos coinjected with sppkc mrna, suggesting that the apoptosis associated with sprunt- deficiency is caused by the deficit in sppkc expression. suppression of the apoptosis by pharmacological inhibition of caspase- prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of sprunt- are secondary to the apoptosis. chromatin immunoprecipitation indicates that sprunt- interacts physically with sppkc in vivo, and cis-regulatory analysis shows that this interaction activates sppkc transcription.
 runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. to discover fundamental runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin runx protein sprunt- previously we showed that embryos depleted of sprunt- arrest development at early gastrula stage and underexpress the conventional protein kinase c sppkc  we report here that sprunt- deficiency leads to ectopic cell proliferation and extensive apoptosis. suppression of the apoptosis by pharmacological inhibition of caspase- prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of sprunt- are secondary to the apoptosis. inhibition or knockdown of sppkc also causes apoptosis, while cell survival is rescued in sprunt- morphant embryos coinjected with sppkc mrna, suggesting that the apoptosis associated with sprunt- deficiency is caused by the deficit in sppkc expression. chromatin immunoprecipitation indicates that sprunt- interacts physically with sppkc in vivo, and cis-regulatory analysis shows that this interaction activates sppkc transcription.  our results show that runx-dependent activation of sppkc is essential for maintaining protein kinase c activity at levels conducive to cell survival during embryogenesis. 