BACKGROUND
for normal homeostasis in multicellular organisms, there exists a delicate balance between cell proliferation and cell death, maintenance of which is required to prevent pathological outcomes including developmental abnormalities, cancer, autoimmune diseases, degenerative disorders and poor outcome following wounds and ischemic injury.
the major physiologic means by which cell death is achieved in an organism is via apoptosis, a tightly regulated and highly conserved process.
in spite of major gains in characterizing the apoptotic pathways in vitro, a better understanding of the precise cellular mechanisms of apoptosis in different tissues and developmental time points in vivo is still required.
the inhibitor of apoptosis proteins  are a family of conserved caspase inhibitors originally identified in baculoviruses as proteins capable of preventing virus-mediated cell death in insect cells.
survivin is the smallest of the human inhibitor of apoptosis proteins , and has several unique features .
it possesses one baculovirus iap repeat  domain that is essential for caspase interference, and contains a c-terminal alpha-helical coiled-coil domain  <cit>  that is important for mitosis.
survivin functions as a chromosome passenger protein, complexing with aurora b, borealin and incenp  <cit> .
survivin also promotes cell cycle progression  <cit> , and is highly expressed by proliferating cells, while being barely detectable in quiescent adult tissues  <cit> .
its uniformly elevated expression in essentially all cancers has rendered survivin a therapeutic target , and thus a critical understanding of the properties of survivin is key for its successful introduction into the clinic.
in both humans and mice, there is a single survivin gene that generates several different protein products due to alternative pre-mrna splicing .
transgenic mouse studies revealed that the major, full-length form of survivin is crucial for normal leukocyte  <cit>  and hepatocyte function  <cit> , hematopoiesis  <cit> , and optimal angiogenic response to injury  <cit> .
elucidating its developmental role has been limited by the fact that survivin gene inactivation in mice causes early embryonic lethality  <cit> .
nonetheless, conditional gene inactivation studies indicate that survivin is essential for brain development  <cit> , angiogenesis and cardiogenesis  <cit> .
in xenopus laevis, there are  <dig> survivin genes, overexpression of one which induces endothelial proliferation  <cit> , while augmented expression of the other had inexplicably lethal effects.
danio rerio  also have  <dig> survivin genes.
ma et al  <cit>  reported that survivin is important in angiogenesis during zebrafish development, but these studies were limited, as the authors did not examine early developmental time points, they restricted their analyses to only one of the genes, and they did not evaluate the importance of survivin in other organ systems.
to gain further insights into the developmental role of survivin, we used the zebrafish model and detailed the spatio-temporal expression patterns of the  <dig> survivin genes and characterized their functions.
zebrafish survivin  <dig>  and survivin  <dig>  both prevent apoptosis and promote cell proliferation.
while they have overlapping patterns of distribution and function, birc5a predominates in most systems.
consistent with its role in the mouse, both zebrafish survivin genes play critical roles in regulating developmental vasculogenesis, angiogenesis, neurogenesis, cardiogenesis, valvulogenesis and hematopoiesis.
the model highlights the conservation of survivin's functions across species, and points to the relevance of utilizing the zebrafish model to evaluate its mechanisms of action, findings that may be extrapolated to human disease.
RESULTS
orthologues of survivin in zebrafish
a blast search for orthologues of human survivin in zebrafish revealed  <dig> survivin genes  located on chromosomes  <dig> and  <dig>  respectively.
using the clustal w program, the putative predicted proteins were aligned with the orthologous proteins found in human, mouse, xenopus laevis and xenopus tropicalis .
human survivin is  <dig> amino acids long, comprised of a  <dig> amino acid n-terminal domain, a bir domain , and a c-terminal coiled-coil domain.
the proteins encoded by birc5a and birc5b exhibit 45–55% overall similarity with those from other species and with each other .
the greatest sequence conservation between birc5a and birc5b resides in the bir domain .
from computer analysis , the c-terminal domains of the zebrafish survivin proteins do not form coiled-coil alpha-helical structures, a region that in murine survivin, interacts with the mitotic spindle.
expression patterns of birc5a and birc5b
ma et al  <cit>  previously described expression of birc5a in  <dig> hpf zebrafish embryos restricted to the developing brain, neural tube, and in cells surrounding the axial vessels.
we performed detailed studies of the expression patterns of both birc5a and birc5b by in situ hybridization using gene-specific probes .
the  <dig> genes entirely overlap in their spatio-temporal patterns of expression.
since there are no prior reports on the expression of birc5b in the zebrafish embryo, we therefore only provide images for birc5b .
birc5a and birc5b were detected as maternal messages throughout the embryo  <dig> hour post fertilization  .
similar to the report by ma et al  <cit>  for birc5a, by 20– <dig> hpf , expression of both genes was prominent in neural tissues, including the entire brain, neural tube, the floor plate, and the midbrain-hindbrain barrier .
in contrast to the report of ma et al, at  <dig> dpf, we also found birc <dig> expression in the ventral somites, at the somite boundaries, and in the caudal vein plexus, and in the eye .
although evident from  <dig> hpf, at  <dig> dpf, there was more prominent expression of both birc <dig> transcripts in the lens and retina of the eyes, in the major axial vessels, and in the branchial arch primordia .
notably, by  <dig> dpf, expression in the somites was almost absent .
by  <dig> dpf, both birc <dig> transcripts were detected in all the branchial arches, in brain, at the midbrain-hindbrain boundary, in the eyes, but was almost entirely absent in the axial vessels and somites .
overall, the expression patterns of the two zebrafish survivin genes are indistinguishable during development, and are predominantly localized to neural, vascular and ocular structures, with transient expression in the somites and axial vessels during their formation.
zebrafish birc <dig> morpholino knockdowns
to assess the functions of birc5a and birc5b, we used morpholinos to knock down each of the genes.
morpholinos were selected to target sites within each orthologue with the most sequence divergence, including the start codon .
specificity of atg morpholinos was confirmed by also using morpholinos that target pre-mrna splice sites or 5' utr regions .
controls were performed by injecting a mismatch morpholino .
each morpholino dose was tested on at least  <dig> embryos.
all morpholinos  for one gene gave identical phenotypes; the results reported reflect those with atg-morpholinos.
we additionally confirmed gene-specificity by in vitro transcription-translation studies.
even at high doses, birc5a morpholinos had no effect on birc5b expression, and similarly, birc5b morpholinos did not affect birc5a expression .
finally, in all studies, we excluded p53-mediated off-target effects on apoptosis and the observed survivin morpholino-induced phenotypes, by co-injecting p <dig> morpholinos, as described  <cit>  .
birc <dig> in neural development
birc5a and birc5b morpholino-injected wild-type ab embryos were assessed by brightfield microscopy .
after injection of  <dig>  ng birc5a morpholino, morphant embryos at  <dig> dpf  and  <dig> dpf  displayed microcephaly  with fluid accumulation in the 4th ventricle .
the effects of birc5b depletion on brain development using the highest morpholino dose were less dramatic than with the birc5a knockdown, with 10–15% of birc5b morphants exhibiting microcephaly at  <dig> dpf  and  <dig> dpf  .
disturbances in neuronal development were, however, more readily detectable in both birc5a and birc5b mutants by in situ hybridization with probes to detect zic <dig>  a pan-neural marker  <cit>  , and islet <dig>  a marker of primary motor neurons  <cit>  .
zic <dig> staining of the brain was decreased in the  <dig> dpf birc5a morphants as compared to controls, reflecting the almost total absence of neural cells.
the reduction was also evident in the birc5b morphants, to a lesser extent when compared to the birc5a morphant.
similarly, islet <dig> staining of the birc5a morphants revealed disorganized or absent motor neurons, while the birc5b morphants were also affected, but again, less severely.
overall, both birc <dig> genes are critical for normal neural development, with birc5a predominating.
birc <dig> in vasculogenesis and angiogenesis
recently, ma et al  <cit>  reported that birc5a knockdowns primarily induce angiogenic abnormalities, but without affecting vasculogenesis.
we extended on their work by examining vasculo-angiogenesis in birc5a and birc5b knockdown embryos at different developmental time points, using tg or tg embryos  which express green fluorescent protein  in endothelial cells .
precursor angioblasts in zebrafish arise at  <dig> hpf  in the lateral plate mesoderm and migrate from  <dig> hpf  toward the midline, where they coalesce to form primary axial vessels  <cit> .
we first established that depletion of birc5a or birc5b does not induce a defect in mesodermal development, by determining that expression of the mesodermal marker, no tail   <cit>  in both morphants at  <dig> hpf, as compared to control zebrafish embryos, was not different .
we then studied the effect of depleting birc5a and birc5b with the highest morpholino doses  on vasculogenesis and angiogenesis.
at  <dig> hpf , birc5a morphant angioblasts migrated in a disorganized fashion, at different rates, with some remaining at the lateral plate mesoderm .
angioblasts in birc5b morphants migrated normally to the midline at  <dig> hpf , but there was reduced signal intensity and thickness of the coalescing axial vessels .
with depletion of birc5a, the dorsal aorta and posterior cardinal vein at  <dig> dpf and  <dig> dpf were thinner , with a smaller caudal vein plexus  .
a similar effect, although not as prominent and not involving the posterior cardinal vein, was also evident in birc5b morphants .
the findings were better visualized following in situ hybridization of  <dig> dpf embryos with probes flt <dig> and gridlock  that detect the posterior cardinal vein and the dorsal aorta/intersomitic vessels, respectively  <cit>  .
the birc5a morphants also exhibited delayed sprouting of intersomitic vessels , which were occasionally absent, but otherwise were often thin, misdirected, lacking connections, and associated with interruption of the dorsal longitudinal anastomotic vessels.
these findings, in concert with the fact that birc <dig> expression was transiently detected at the somite boundaries, suggests a role for survivin in isv patterning and vessel guidance .
interestingly and in contrast, formation of the intersomitic vessels and dorsal longitudinal anastomotic vessels remained intact in the birc5b morphants.
by  <dig> dpf, there was underdevelopment and irregular patterning of cranial blood vessels of both the birc5a and birc5b morphants  as compared with controls.
only  <dig> of the branchial arches was seen in the majority of the birc5a morphants  , while the 5th and 6th branchial arches in the birc5b morphants were either absent or hypoplastic   .
in attempting to explain the branchial arch defects, we performed in situ hybridization studies at  <dig> dpf with crestin, a post migratory neural crest cell marker  <cit> .
in both the birc5a and the birc5b morphants, but more prominent with depletion of birc5a, there were fewer crestin-positive neural crest cells in the region of the neural crest, corresponding to that which is critical for development of the branchial arches  <cit>  .
in summary, both birc <dig> genes are important for normal vasculogenesis, angiogenesis, and vascular patterning.
although the  <dig> genes functionally overlap, again, birc5a appears to play a more prominent role.
birc <dig> in hematopoiesis
since, in the zebrafish, at least a subset of hematopoietic and endothelial lineages arise from a common hemangioblast  <cit> , we also examined birc5a and birc5b morphants for defective hematopoiesis.
erythrocytes in  <dig> dpf embryos were identified by staining for hemoglobin with o-dianisidine.
depletion of birc5a or birc5b resulted in a reduced number of erythrocytes in the ducts of cuvier .
moreover, gata <dig> expression, reflecting hematopoiesis at  <dig> dpf, was reduced in 35% of birc5a morphants , and 28% of birc5b morphants , as compared to controls  .
since defects in circulation may impact on the preceding findings, we examined hematopoiesis at  <dig> hpf, prior to development of a functional circulation.
flow cytometry of cellular suspensions of dechorianated tg embryos was used to quantify the number of gata1+ cells relative to the total number of cells  <cit> .
depletion of either birc5a or birc5b, as compared to control injections, resulted in a decrease in the absolute number of gata1+ cells from  <dig> % for controls to  <dig> % for birc5a, and to  <dig> % for birc5b.
in line with these findings, expression of hematopoietic genes gata <dig>  scl and imo <dig>  <cit> , quantified by real-time pcr at  <dig> hpf and  <dig> hpf in ab zebrafish embryos, was significantly reduced  by depletion of either birc5a or birc5b.
overall, the data support a role for both birc <dig> genes in promoting hematopoiesis in the developing zebrafish embryo.
birc <dig> in cardiogenesis
depletion of either birc5a or birc5b resulted in cardiovascular defects.
at the highest morpholino dose, one third of birc5a morphants displayed pericardial edema, while blood flow in the dorsal aorta and posterior cardinal vein was slowed or absent in at least half the embryos.
the heart rate in both birc5a and birc5b morphants was significantly reduced .
in situ hybridization at  <dig> hpf with the ventricle and cardiac-specific markers, ventricle myosin heavy chain  and cardiac myosin light chain   <cit> , respectively, demonstrated that birc5a morphant ventricles and hearts were smaller than controls  .
although not as striking, birc5b morphants also exhibited smaller ventricles and hearts at  <dig> hpf  .
at  <dig> hpf when atrio-ventricular  valve formation is underway, bone morphogenic protein  <dig>   <cit>  transcripts progressively localize from the atrium and ventricle to myocardial cells at the valve-forming region  <cit> .
with birc5a depletion, redistribution of bmp <dig> did not occur in 38%  of embryos , predicting defects in valvulogenesis.
this was evident in hematoxylin and eosin  stained sections of  <dig> dpf embryos, where there was little evidence of an a-v valve in the birc5a morphants, at a time when the myocardial layer of the morphant ventricle was also thinner, as compared to control embryos .
in the birc5b morphants, bmp <dig> became localized to the a-v valve, similar to controls, although the opening was smaller in 21%  .
at  <dig> dpf, h&e stained sections also revealed smaller cardiac chambers  .
although diminished vascular perfusion may contribute to cardiogenic defects  <cit> , we hypothesized that an additional factor in the birc <dig> morphants might be altered formation and/or migration of cardiac neural crest cells, a site of origin of cardiomyocytes in zebrafish  <cit> .
embryos at  <dig>  hpf  were therefore hybridized with the foxd <dig> probe to stain premigratory neural crest cells  <cit> .
birc5a depletion caused cardiac neural crest cell loss along the rostrocaudal axis , particularly in the region critical for formation of the ventricle, a-v junction, and atrium .
birc5b depletion also resulted in loss of cardiac neural crest cells – again, not as dramatically as with birc5a depletion .
overall, the findings indicate that birc5a- and birc5b-dependent signals are important in maintaining the integrity of cardiac neural crest cells that in turn, contribute to normal cardiogenesis and valvulogenesis.
birc <dig> knockdowns result in increased apoptosis and decreased cell proliferation
ma et al  <cit>  reported that depletion of birc5a caused increased apoptosis primarily in the neural tube and brain.
given the diverse pro-survival properties of survivin, we assessed the mechanisms of action of each zebrafish survivin gene in the neural and vascular systems.
tunel staining of normal tg embryos at  <dig> dpf  and  <dig> dpf  revealed minimal evidence of apoptosis.
in contrast, at a morpholino dose of  <dig> ng, birc5a morphants exhibited significant apoptosis, mostly in the brain and spinal cord , with lesser amounts in the region of the axial vessels and caudal vein plexus .
tunel staining in the birc5b morphants at  <dig> dpf was, by comparison, less in the neural tissues, but more prominent in the region of the caudal vein plexus and axial vessels .
with both gene knockdowns, apoptosis remained spatially unchanged, but was increased at  <dig> dpf .
quantification of tunel positive cells in the caudal vein plexus and the corresponding dorsal neural tube region confirmed that depletion of either birc5a or birc5b at the higher dose, caused a significant increase in the number of apoptotic cells in both regions  .
however, when the birc5a morpholino dose was decreased to  <dig>  ng, apoptosis in the caudal vein plexus entirely resolved, while tunel staining in the neural tube persisted , indicating a greater sensitivity of the neural structures to loss of birc5a.
conversely, as the birc5b morpholino dose was decreased, the effect on the neural tube diminished, while apoptosis in the region of the caudal vein plexus persisted .
we also quantified the effects of the two genes on cell proliferation .
at  <dig> dpf, proliferating cells were present in the region of the caudal vein plexus and neural tube .
birc5a depletion using  <dig> ng of morpholino interfered with cell proliferation in both regions, while the  <dig>  ng dose  only suppressed proliferation in the neural tube.
depletion of birc5b also interfered with cell proliferation in the caudal vein plexus and neural tube, and this anti-proliferative effect in the neural tube disappeared at the lower morpholino dose .
overall, the findings support the notion that both birc <dig> genes interfere with apoptosis and promote cell proliferation during early zebrafish development, but that there are dose-dependent and site-specific distinguishing features.
phenotype rescues of birc5a and birc5b morphants
we determined whether we could rescue the birc <dig> morphants by co-injecting synthetic mrnas encoding the open reading frame of either birc5a or birc5b.
in a dose-dependent manner, co-injection of the respective birc <dig> mrna almost entirely rescued both the vascular and neural phenotypes induced by the highest morpholino dose .
thus, at  <dig> dpf, the birc5a morphants were rescued by co-injection of  <dig> ng of birc5a mrna, and the birc5b morphants were rescued by  <dig> ng of birc5b mrna.
furthermore, co-injection of  <dig> ng of birc5b mrna could also partially rescue the birc5a morphants, while birc5b morphants could be completely rescued by birc5a mrna.
the findings indicate that the  <dig> survivin genes may, under different conditions, compensate for each other.
birc <dig> morpholinos were injected into tg zebrafish embryos simultaneously with injections of specific mrnas as shown, and phenotypic analyses were performed at  <dig> dpf.
the incidence of neural or vascular abnormalities is illustrated.
role of vegf in regulating survivin
vascular endothelial growth factor  has vasculo-angiogenic, neurogenic, cardiogenic and hematopoietic properties, the effects on endothelial cells mediated in part by upregulating survivin  <cit> .
ma et al  <cit>  demonstrated that vegf protein upregulates expression of birc5a in zebrafish at  <dig> hpf.
we evaluated whether vegf could rescue the phenotypes induced by depletion of the  <dig> zebrafish survivin genes.
human vegf mrna  or vehicle was injected into tg embryos alone, or with a control morpholino, or with maximum dose morpholinos to deplete either birc5a or birc5b .
embryos were evaluated at  <dig> dpf.
in conjunction with birc5a morpholino knockdowns , vegf mrna reduced the incidence of neural and vascular defects to 43% and 37%, respectively, of that observed with birc5a morpholino alone.
vegf also rescued the phenotype induced by birc5b depletion.
thus, administration of vegf mrna  with birc5b morpholino decreased the incidence of vascular and neural defects in birc5b morphants , to 20% and 24%, respectively, of that found with birc5b morpholino alone.
the results demonstrate that vegf may protect the integrity of the neural and vascular systems from single birc <dig> gene depletion, possibly via a compensatory increase in expression of the second birc <dig> gene.
discussion and
CONCLUSIONS
in attempting to elucidate the physiologic relevance of the inhibitor of apoptosis protein, survivin, we have utilized the zebrafish model and characterized the expression patterns and functions of its two genes in early development.
our studies extend those of ma et al  <cit> , who first reported that birc5a has angiogenic, but not vasculogenic, properties, a discrepancy that may be partly explained by the fact that their studies were restricted to developmental time points no earlier than  <dig> hpf.
several novel insights are provided by our work.
both birc5a and birc5b are expressed predominantly by neural, vascular, and ocular structures, and in the somites/somite boundaries; both inhibit apoptosis and promote cell proliferation; and both contribute to normal vasculogenesis, angiogenesis, cardiogenesis, neurogenesis and hematopoiesis.
no other member of the iap family has been shown to have similarly profound developmental effects on multiple organ systems following gene inactivation or knockdown in small animal models  <cit> .
our findings are consistent with immunohistochemical analyses and survivin gene inactivation studies in the mouse embryo, where the single survivin gene is essential for survival, and also plays a key role in angiogenesis, neurogenesis, cardiogenesis and hematopoiesis  <cit> .
our studies therefore support the concept that the zebrafish paralogs, birc5a and birc5b, largely recapitulate the properties of the ancestral gene, represented in the mouse, thereby rationalizing the use of this model to elucidate the role of survivin in health and disease in higher animals.
while the two zebrafish survivin genes have indistinguishable patterns of expression during development, in all of our assays, except those for hematopoiesis, depletion of birc5a resulted in more severe, and sometimes distinct phenotypes.
for example, only birc5a depletion caused marked alterations in isv patterning.
this phenotype would be most readily attributed to expression of birc5a at the somite boundaries, where vegf  <cit>  and other guidance molecules play a crucial role in vessel patterning .
however, birc5b is also expressed in the somites/somite boundaries, and its depletion had no effect on the isvs.
thus, further studies, including analyses of the expression profiles of relevant guidance molecules, will be required to further elucidate the specific properties of each birc <dig> gene in the somites.
functional differences between the two birc <dig> genes were additionally uncovered by titering the respective morpholino doses to evaluate effects of each paralog on apoptosis and cell proliferation in the neural tube and vascular structures.
by this approach, birc5a was found to be more effective at protecting the neural structures, while birc5b was more effective at protecting the caudal vein plexus.
the latter is interesting, because the caudal vein plexus is a major site for primitive hematopoiesis, a process that was more prominently disturbed by birc5b depletion.
nonetheless, administration of either mrna could effectively rescue the phenotypes induced by depletion of the other.
thus, the physiologic relevance of the functional differences between the two survivin paralogs is not fully delineated, and it appears that each may act to compensate for deficiencies of the other.
a common feature of both birc <dig> genes, not previously recognized, is that their expression in the somites and axial vessels is transient and restricted to an early developmental time period, both essentially gone by  <dig> dpf.
up until that time, the axial vessels and the isvs are generated, and based on the morpholino knockdowns, survivin is critical for formation of these structures, after which survivin is no longer necessary at those sites.
interestingly, the iap birc <dig> is expressed in the vasculature of zebrafish beginning at  <dig> hpf, whereupon it is required to maintain endothelial cell integrity  <cit> .
one could speculate that there exists an intrinsic molecular switch that is "flipped" at 2– <dig> dpf, when birc <dig> expression turns off, and birc <dig> turns on, the latter which is required to form a more complex vascular network.
characterization of such a switch mechanism could enhance our understanding of the regulation of angiogenesis.
in mice depleted of endothelial survivin, embryonic heart development was abnormal, and the mutant endocardial lineage cells could not support epithelial-mesenchymal transformation   <cit> .
in both survivin gene knockdowns in the zebrafish, we also observed abnormalities in cardiogenesis – more prominent with birc5a depletion.
fate-mapping studies in zebrafish have revealed that formation of the atrium, ventricle and a-v valves depends on the integrity of the cardiac neural crest cells  <cit> , while intracardiac fluid forces also contribute to normal heart development  <cit> .
thus, the etiology of the abnormalities in cardiogenesis in the birc <dig> morphants may be multifactorial, i.e.
secondary to the circulation defect and/or due to loss of the cardiac neural crest cells.
further study is required to elucidate the relevant birc5-dependent pro-survival pathways for these neural crest cells.
nonetheless, the zebrafish and mouse models highlight the importance of survivin in heart development.
just as single-nucleotide polymorphisms of the vegf gene have been linked to congenital valvuloseptal defects  <cit> , the possibility that functional alterations in survivin expression might underlie congenital heart defects is worthy of consideration.
the prominent role that survivin plays in regulating vasculo-angiogenesis, neurogenesis, cardiogenesis and hematopoiesis supports the widely accepted notion of co-ordinately regulated development of these systems .
for example, the eph/ephrins regulate fasciculation and guidance of axons, direct neural crest cell fate and migration, modulate neural progenitor cell survival, while also being important for cardiovascular development  <cit>  and erythropoiesis  <cit> .
vascular endothelial growth factor , although best characterized as a critical mediator of vasculogenesis and angiogenesis, also has direct effects on the nervous system .
vegf protects neural cells from hypoxia, facilitates axonal outgrowth, promotes endothelial release of neurogenic factors  <cit> , and induces neural stem cell proliferation  <cit> .
vegf also promotes hematopoiesis  <cit> , and altered regulation of vegf results in profound defects in heart development  <cit> .
these properties of vegf are confirmed in our studies, where the neural and cardiovascular phenotypes induced by depletion of birc5a or birc5b, were rescued by vegf.
although not tested, it is likely that upon depletion of one birc <dig> gene, the exogenous vegf upregulated expression of the other, which in turn compensated for the phenotypic defects.
in that respect, ma et al  <cit>  indeed, demonstrated that vegf protein can increase accumulation of birc5a mrna in zebrafish embryos.
beyond the zebrafish model system, upregulation of survivin in endothelial cells has been well-documented  <cit> .
the finding that survivin is also a downstream effector of vegf in the neurologic system, and that survivin transcripts are highly expressed in neural progenitor cells  <cit> , implies that the survivin gene in humans may, similar to vegf  <cit> , be a modifier in the progression of neural diseases, such as amyotrophic lateral sclerosis, and thus have the potential as a therapeutic target.
by regulating cell proliferation and apoptosis, the two zebrafish survivin genes, birc5a and birc5b, play important roles in multiple biologic systems.
although we have not examined all organs during development; nor have we yet evaluated its role in the eye; the prominent effects of birc <dig> gene depletion on the cardiac, neurovascular and hematopoietic systems in the zebrafish embryo suggest that survivin has temporal and tissue-specific properties.
in view of the apparent functional overlap with the murine and human survivin orthologues, the zebrafish model provides an exceptional opportunity to examine the physiologic relevance of the complex molecular and biochemical pathways that govern cell survival and apoptosis.
the insights gained will lead to the development of safer, targeted therapeutics for a spectrum of cardio-vascular, hematopoeitic and neurologic diseases, and a better understanding of the etiology and genetics of an array of congenital diseases.

 for normal homeostasis in multicellular organisms, there exists a delicate balance between cell proliferation and cell death, maintenance of which is required to prevent pathological outcomes including developmental abnormalities, cancer, autoimmune diseases, degenerative disorders and poor outcome following wounds and ischemic injury. the major physiologic means by which cell death is achieved in an organism is via apoptosis, a tightly regulated and highly conserved process. in spite of major gains in characterizing the apoptotic pathways in vitro, a better understanding of the precise cellular mechanisms of apoptosis in different tissues and developmental time points in vivo is still required. the inhibitor of apoptosis proteins are a family of conserved caspase inhibitors originally identified in baculoviruses as proteins capable of preventing virus-mediated cell death in insect cells. survivin is the smallest of the human inhibitor of apoptosis proteins, and has several unique features. it possesses one baculovirus iap repeat domain that is essential for caspase interference, and contains a c-terminal alpha-helical coiled-coil domain that is important for mitosis. survivin functions as a chromosome passenger protein, complexing with aurora b, borealin and incenp. survivin also promotes cell cycle progression, and is highly expressed by proliferating cells, while being barely detectable in quiescent adult tissues. its uniformly elevated expression in essentially all cancers has rendered survivin a therapeutic target, and thus a critical understanding of the properties of survivin is key for its successful introduction into the clinic. in both humans and mice, there is a single survivin gene that generates several different protein products due to alternative pre-mrna splicing. transgenic mouse studies revealed that the major, full-length form of survivin is crucial for normal leukocyte and hepatocyte function, hematopoiesis, and optimal angiogenic response to injury. elucidating its developmental role has been limited by the fact that survivin gene inactivation in mice causes early embryonic lethality. nonetheless, conditional gene inactivation studies indicate that survivin is essential for brain development, angiogenesis and cardiogenesis. in xenopus laevis, there are survivin genes, overexpression of one which induces endothelial proliferation, while augmented expression of the other had inexplicably lethal effects. danio rerio also have survivin genes. ma et al reported that survivin is important in angiogenesis during zebrafish development, but these studies were limited, as the authors did not examine early developmental time points, they restricted their analyses to only one of the genes, and they did not evaluate the importance of survivin in other organ systems. to gain further insights into the developmental role of survivin, we used the zebrafish model and detailed the spatio-temporal expression patterns of the survivin genes and characterized their functions. zebrafish survivin and survivin both prevent apoptosis and promote cell proliferation. while they have overlapping patterns of distribution and function, birc5a predominates in most systems. consistent with its role in the mouse, both zebrafish survivin genes play critical roles in regulating developmental vasculogenesis, angiogenesis, neurogenesis, cardiogenesis, valvulogenesis and hematopoiesis. the model highlights the conservation of survivin's functions across species, and points to the relevance of utilizing the zebrafish model to evaluate its mechanisms of action, findings that may be extrapolated to human disease.  orthologues of survivin in zebrafish a blast search for orthologues of human survivin in zebrafish revealed survivin genes located on chromosomes and respectively. using the clustal w program, the putative predicted proteins were aligned with the orthologous proteins found in human, mouse, xenopus laevis and xenopus tropicalis. human survivin is amino acids long, comprised of a amino acid n-terminal domain, a bir domain, and a c-terminal coiled-coil domain. the proteins encoded by birc5a and birc5b exhibit 45–55% overall similarity with those from other species and with each other. the greatest sequence conservation between birc5a and birc5b resides in the bir domain. from computer analysis, the c-terminal domains of the zebrafish survivin proteins do not form coiled-coil alpha-helical structures, a region that in murine survivin, interacts with the mitotic spindle. expression patterns of birc5a and birc5b ma et al previously described expression of birc5a in hpf zebrafish embryos restricted to the developing brain, neural tube, and in cells surrounding the axial vessels. we performed detailed studies of the expression patterns of both birc5a and birc5b by in situ hybridization using gene-specific probes. the genes entirely overlap in their spatio-temporal patterns of expression. since there are no prior reports on the expression of birc5b in the zebrafish embryo, we therefore only provide images for birc5b. birc5a and birc5b were detected as maternal messages throughout the embryo hour post fertilization. similar to the report by ma et al for birc5a, by 20– hpf, expression of both genes was prominent in neural tissues, including the entire brain, neural tube, the floor plate, and the midbrain-hindbrain barrier. in contrast to the report of ma et al, at dpf, we also found birc expression in the ventral somites, at the somite boundaries, and in the caudal vein plexus, and in the eye. although evident from hpf, at dpf, there was more prominent expression of both birc transcripts in the lens and retina of the eyes, in the major axial vessels, and in the branchial arch primordia. notably, by dpf, expression in the somites was almost absent. by dpf, both birc transcripts were detected in all the branchial arches, in brain, at the midbrain-hindbrain boundary, in the eyes, but was almost entirely absent in the axial vessels and somites. overall, the expression patterns of the two zebrafish survivin genes are indistinguishable during development, and are predominantly localized to neural, vascular and ocular structures, with transient expression in the somites and axial vessels during their formation. zebrafish birc morpholino knockdowns to assess the functions of birc5a and birc5b, we used morpholinos to knock down each of the genes. morpholinos were selected to target sites within each orthologue with the most sequence divergence, including the start codon. specificity of atg morpholinos was confirmed by also using morpholinos that target pre-mrna splice sites or 5' utr regions. controls were performed by injecting a mismatch morpholino. each morpholino dose was tested on at least embryos. all morpholinos for one gene gave identical phenotypes; the results reported reflect those with atg-morpholinos. we additionally confirmed gene-specificity by in vitro transcription-translation studies. even at high doses, birc5a morpholinos had no effect on birc5b expression, and similarly, birc5b morpholinos did not affect birc5a expression. finally, in all studies, we excluded p53-mediated off-target effects on apoptosis and the observed survivin morpholino-induced phenotypes, by co-injecting p morpholinos, as described. birc in neural development birc5a and birc5b morpholino-injected wild-type ab embryos were assessed by brightfield microscopy. after injection of ng birc5a morpholino, morphant embryos at dpf and dpf displayed microcephaly with fluid accumulation in the 4th ventricle. the effects of birc5b depletion on brain development using the highest morpholino dose were less dramatic than with the birc5a knockdown, with 10–15% of birc5b morphants exhibiting microcephaly at dpf and dpf. disturbances in neuronal development were, however, more readily detectable in both birc5a and birc5b mutants by in situ hybridization with probes to detect zic a pan-neural marker, and islet a marker of primary motor neurons. zic staining of the brain was decreased in the dpf birc5a morphants as compared to controls, reflecting the almost total absence of neural cells. the reduction was also evident in the birc5b morphants, to a lesser extent when compared to the birc5a morphant. similarly, islet staining of the birc5a morphants revealed disorganized or absent motor neurons, while the birc5b morphants were also affected, but again, less severely. overall, both birc genes are critical for normal neural development, with birc5a predominating. birc in vasculogenesis and angiogenesis recently, ma et al reported that birc5a knockdowns primarily induce angiogenic abnormalities, but without affecting vasculogenesis. we extended on their work by examining vasculo-angiogenesis in birc5a and birc5b knockdown embryos at different developmental time points, using tg or tg embryos which express green fluorescent protein in endothelial cells. precursor angioblasts in zebrafish arise at hpf in the lateral plate mesoderm and migrate from hpf toward the midline, where they coalesce to form primary axial vessels. we first established that depletion of birc5a or birc5b does not induce a defect in mesodermal development, by determining that expression of the mesodermal marker, no tail in both morphants at hpf, as compared to control zebrafish embryos, was not different. we then studied the effect of depleting birc5a and birc5b with the highest morpholino doses on vasculogenesis and angiogenesis. at hpf, birc5a morphant angioblasts migrated in a disorganized fashion, at different rates, with some remaining at the lateral plate mesoderm. angioblasts in birc5b morphants migrated normally to the midline at hpf, but there was reduced signal intensity and thickness of the coalescing axial vessels. with depletion of birc5a, the dorsal aorta and posterior cardinal vein at dpf and dpf were thinner, with a smaller caudal vein plexus. a similar effect, although not as prominent and not involving the posterior cardinal vein, was also evident in birc5b morphants. the findings were better visualized following in situ hybridization of dpf embryos with probes flt and gridlock that detect the posterior cardinal vein and the dorsal aorta/intersomitic vessels, respectively. the birc5a morphants also exhibited delayed sprouting of intersomitic vessels, which were occasionally absent, but otherwise were often thin, misdirected, lacking connections, and associated with interruption of the dorsal longitudinal anastomotic vessels. these findings, in concert with the fact that birc expression was transiently detected at the somite boundaries, suggests a role for survivin in isv patterning and vessel guidance. interestingly and in contrast, formation of the intersomitic vessels and dorsal longitudinal anastomotic vessels remained intact in the birc5b morphants. by dpf, there was underdevelopment and irregular patterning of cranial blood vessels of both the birc5a and birc5b morphants as compared with controls. only of the branchial arches was seen in the majority of the birc5a morphants, while the 5th and 6th branchial arches in the birc5b morphants were either absent or hypoplastic. in attempting to explain the branchial arch defects, we performed in situ hybridization studies at dpf with crestin, a post migratory neural crest cell marker. in both the birc5a and the birc5b morphants, but more prominent with depletion of birc5a, there were fewer crestin-positive neural crest cells in the region of the neural crest, corresponding to that which is critical for development of the branchial arches. in summary, both birc genes are important for normal vasculogenesis, angiogenesis, and vascular patterning. although the genes functionally overlap, again, birc5a appears to play a more prominent role. birc in hematopoiesis since, in the zebrafish, at least a subset of hematopoietic and endothelial lineages arise from a common hemangioblast, we also examined birc5a and birc5b morphants for defective hematopoiesis. erythrocytes in dpf embryos were identified by staining for hemoglobin with o-dianisidine. depletion of birc5a or birc5b resulted in a reduced number of erythrocytes in the ducts of cuvier. moreover, gata expression, reflecting hematopoiesis at dpf, was reduced in 35% of birc5a morphants, and 28% of birc5b morphants, as compared to controls. since defects in circulation may impact on the preceding findings, we examined hematopoiesis at hpf, prior to development of a functional circulation. flow cytometry of cellular suspensions of dechorianated tg embryos was used to quantify the number of gata1+ cells relative to the total number of cells. depletion of either birc5a or birc5b, as compared to control injections, resulted in a decrease in the absolute number of gata1+ cells from % for controls to % for birc5a, and to % for birc5b. in line with these findings, expression of hematopoietic genes gata scl and imo, quantified by real-time pcr at hpf and hpf in ab zebrafish embryos, was significantly reduced by depletion of either birc5a or birc5b. overall, the data support a role for both birc genes in promoting hematopoiesis in the developing zebrafish embryo. birc in cardiogenesis depletion of either birc5a or birc5b resulted in cardiovascular defects. at the highest morpholino dose, one third of birc5a morphants displayed pericardial edema, while blood flow in the dorsal aorta and posterior cardinal vein was slowed or absent in at least half the embryos. the heart rate in both birc5a and birc5b morphants was significantly reduced. in situ hybridization at hpf with the ventricle and cardiac-specific markers, ventricle myosin heavy chain and cardiac myosin light chain, respectively, demonstrated that birc5a morphant ventricles and hearts were smaller than controls. although not as striking, birc5b morphants also exhibited smaller ventricles and hearts at hpf. at hpf when atrio-ventricular valve formation is underway, bone morphogenic protein transcripts progressively localize from the atrium and ventricle to myocardial cells at the valve-forming region. with birc5a depletion, redistribution of bmp did not occur in 38% of embryos, predicting defects in valvulogenesis. this was evident in hematoxylin and eosin stained sections of dpf embryos, where there was little evidence of an a-v valve in the birc5a morphants, at a time when the myocardial layer of the morphant ventricle was also thinner, as compared to control embryos. in the birc5b morphants, bmp became localized to the a-v valve, similar to controls, although the opening was smaller in 21%. at dpf, h&e stained sections also revealed smaller cardiac chambers. although diminished vascular perfusion may contribute to cardiogenic defects, we hypothesized that an additional factor in the birc morphants might be altered formation and/or migration of cardiac neural crest cells, a site of origin of cardiomyocytes in zebrafish. embryos at hpf were therefore hybridized with the foxd probe to stain premigratory neural crest cells. birc5a depletion caused cardiac neural crest cell loss along the rostrocaudal axis, particularly in the region critical for formation of the ventricle, a-v junction, and atrium. birc5b depletion also resulted in loss of cardiac neural crest cells – again, not as dramatically as with birc5a depletion. overall, the findings indicate that birc5a- and birc5b-dependent signals are important in maintaining the integrity of cardiac neural crest cells that in turn, contribute to normal cardiogenesis and valvulogenesis. birc knockdowns result in increased apoptosis and decreased cell proliferation ma et al reported that depletion of birc5a caused increased apoptosis primarily in the neural tube and brain. given the diverse pro-survival properties of survivin, we assessed the mechanisms of action of each zebrafish survivin gene in the neural and vascular systems. tunel staining of normal tg embryos at dpf and dpf revealed minimal evidence of apoptosis. in contrast, at a morpholino dose of ng, birc5a morphants exhibited significant apoptosis, mostly in the brain and spinal cord, with lesser amounts in the region of the axial vessels and caudal vein plexus. tunel staining in the birc5b morphants at dpf was, by comparison, less in the neural tissues, but more prominent in the region of the caudal vein plexus and axial vessels. with both gene knockdowns, apoptosis remained spatially unchanged, but was increased at dpf. quantification of tunel positive cells in the caudal vein plexus and the corresponding dorsal neural tube region confirmed that depletion of either birc5a or birc5b at the higher dose, caused a significant increase in the number of apoptotic cells in both regions. however, when the birc5a morpholino dose was decreased to ng, apoptosis in the caudal vein plexus entirely resolved, while tunel staining in the neural tube persisted, indicating a greater sensitivity of the neural structures to loss of birc5a. conversely, as the birc5b morpholino dose was decreased, the effect on the neural tube diminished, while apoptosis in the region of the caudal vein plexus persisted. we also quantified the effects of the two genes on cell proliferation. at dpf, proliferating cells were present in the region of the caudal vein plexus and neural tube. birc5a depletion using ng of morpholino interfered with cell proliferation in both regions, while the ng dose only suppressed proliferation in the neural tube. depletion of birc5b also interfered with cell proliferation in the caudal vein plexus and neural tube, and this anti-proliferative effect in the neural tube disappeared at the lower morpholino dose. overall, the findings support the notion that both birc genes interfere with apoptosis and promote cell proliferation during early zebrafish development, but that there are dose-dependent and site-specific distinguishing features. phenotype rescues of birc5a and birc5b morphants we determined whether we could rescue the birc morphants by co-injecting synthetic mrnas encoding the open reading frame of either birc5a or birc5b. in a dose-dependent manner, co-injection of the respective birc mrna almost entirely rescued both the vascular and neural phenotypes induced by the highest morpholino dose. thus, at dpf, the birc5a morphants were rescued by co-injection of ng of birc5a mrna, and the birc5b morphants were rescued by ng of birc5b mrna. furthermore, co-injection of ng of birc5b mrna could also partially rescue the birc5a morphants, while birc5b morphants could be completely rescued by birc5a mrna. the findings indicate that the survivin genes may, under different conditions, compensate for each other. birc morpholinos were injected into tg zebrafish embryos simultaneously with injections of specific mrnas as shown, and phenotypic analyses were performed at dpf. the incidence of neural or vascular abnormalities is illustrated. role of vegf in regulating survivin vascular endothelial growth factor has vasculo-angiogenic, neurogenic, cardiogenic and hematopoietic properties, the effects on endothelial cells mediated in part by upregulating survivin. ma et al demonstrated that vegf protein upregulates expression of birc5a in zebrafish at hpf. we evaluated whether vegf could rescue the phenotypes induced by depletion of the zebrafish survivin genes. human vegf mrna or vehicle was injected into tg embryos alone, or with a control morpholino, or with maximum dose morpholinos to deplete either birc5a or birc5b. embryos were evaluated at dpf. in conjunction with birc5a morpholino knockdowns, vegf mrna reduced the incidence of neural and vascular defects to 43% and 37%, respectively, of that observed with birc5a morpholino alone. vegf also rescued the phenotype induced by birc5b depletion. thus, administration of vegf mrna with birc5b morpholino decreased the incidence of vascular and neural defects in birc5b morphants, to 20% and 24%, respectively, of that found with birc5b morpholino alone. the results demonstrate that vegf may protect the integrity of the neural and vascular systems from single birc gene depletion, possibly via a compensatory increase in expression of the second birc gene. discussion and  in attempting to elucidate the physiologic relevance of the inhibitor of apoptosis protein, survivin, we have utilized the zebrafish model and characterized the expression patterns and functions of its two genes in early development. our studies extend those of ma et al, who first reported that birc5a has angiogenic, but not vasculogenic, properties, a discrepancy that may be partly explained by the fact that their studies were restricted to developmental time points no earlier than hpf. several novel insights are provided by our work. both birc5a and birc5b are expressed predominantly by neural, vascular, and ocular structures, and in the somites/somite boundaries; both inhibit apoptosis and promote cell proliferation; and both contribute to normal vasculogenesis, angiogenesis, cardiogenesis, neurogenesis and hematopoiesis. no other member of the iap family has been shown to have similarly profound developmental effects on multiple organ systems following gene inactivation or knockdown in small animal models. our findings are consistent with immunohistochemical analyses and survivin gene inactivation studies in the mouse embryo, where the single survivin gene is essential for survival, and also plays a key role in angiogenesis, neurogenesis, cardiogenesis and hematopoiesis. our studies therefore support the concept that the zebrafish paralogs, birc5a and birc5b, largely recapitulate the properties of the ancestral gene, represented in the mouse, thereby rationalizing the use of this model to elucidate the role of survivin in health and disease in higher animals. while the two zebrafish survivin genes have indistinguishable patterns of expression during development, in all of our assays, except those for hematopoiesis, depletion of birc5a resulted in more severe, and sometimes distinct phenotypes. for example, only birc5a depletion caused marked alterations in isv patterning. this phenotype would be most readily attributed to expression of birc5a at the somite boundaries, where vegf and other guidance molecules play a crucial role in vessel patterning. however, birc5b is also expressed in the somites/somite boundaries, and its depletion had no effect on the isvs. thus, further studies, including analyses of the expression profiles of relevant guidance molecules, will be required to further elucidate the specific properties of each birc gene in the somites. functional differences between the two birc genes were additionally uncovered by titering the respective morpholino doses to evaluate effects of each paralog on apoptosis and cell proliferation in the neural tube and vascular structures. by this approach, birc5a was found to be more effective at protecting the neural structures, while birc5b was more effective at protecting the caudal vein plexus. the latter is interesting, because the caudal vein plexus is a major site for primitive hematopoiesis, a process that was more prominently disturbed by birc5b depletion. nonetheless, administration of either mrna could effectively rescue the phenotypes induced by depletion of the other. thus, the physiologic relevance of the functional differences between the two survivin paralogs is not fully delineated, and it appears that each may act to compensate for deficiencies of the other. a common feature of both birc genes, not previously recognized, is that their expression in the somites and axial vessels is transient and restricted to an early developmental time period, both essentially gone by dpf. up until that time, the axial vessels and the isvs are generated, and based on the morpholino knockdowns, survivin is critical for formation of these structures, after which survivin is no longer necessary at those sites. interestingly, the iap birc is expressed in the vasculature of zebrafish beginning at hpf, whereupon it is required to maintain endothelial cell integrity. one could speculate that there exists an intrinsic molecular switch that is "flipped" at 2– dpf, when birc expression turns off, and birc turns on, the latter which is required to form a more complex vascular network. characterization of such a switch mechanism could enhance our understanding of the regulation of angiogenesis. in mice depleted of endothelial survivin, embryonic heart development was abnormal, and the mutant endocardial lineage cells could not support epithelial-mesenchymal transformation. in both survivin gene knockdowns in the zebrafish, we also observed abnormalities in cardiogenesis – more prominent with birc5a depletion. fate-mapping studies in zebrafish have revealed that formation of the atrium, ventricle and a-v valves depends on the integrity of the cardiac neural crest cells, while intracardiac fluid forces also contribute to normal heart development. thus, the etiology of the abnormalities in cardiogenesis in the birc morphants may be multifactorial, i.e. secondary to the circulation defect and/or due to loss of the cardiac neural crest cells. further study is required to elucidate the relevant birc5-dependent pro-survival pathways for these neural crest cells. nonetheless, the zebrafish and mouse models highlight the importance of survivin in heart development. just as single-nucleotide polymorphisms of the vegf gene have been linked to congenital valvuloseptal defects, the possibility that functional alterations in survivin expression might underlie congenital heart defects is worthy of consideration. the prominent role that survivin plays in regulating vasculo-angiogenesis, neurogenesis, cardiogenesis and hematopoiesis supports the widely accepted notion of co-ordinately regulated development of these systems. for example, the eph/ephrins regulate fasciculation and guidance of axons, direct neural crest cell fate and migration, modulate neural progenitor cell survival, while also being important for cardiovascular development and erythropoiesis. vascular endothelial growth factor, although best characterized as a critical mediator of vasculogenesis and angiogenesis, also has direct effects on the nervous system. vegf protects neural cells from hypoxia, facilitates axonal outgrowth, promotes endothelial release of neurogenic factors, and induces neural stem cell proliferation. vegf also promotes hematopoiesis, and altered regulation of vegf results in profound defects in heart development. these properties of vegf are confirmed in our studies, where the neural and cardiovascular phenotypes induced by depletion of birc5a or birc5b, were rescued by vegf. although not tested, it is likely that upon depletion of one birc gene, the exogenous vegf upregulated expression of the other, which in turn compensated for the phenotypic defects. in that respect, ma et al indeed, demonstrated that vegf protein can increase accumulation of birc5a mrna in zebrafish embryos. beyond the zebrafish model system, upregulation of survivin in endothelial cells has been well-documented. the finding that survivin is also a downstream effector of vegf in the neurologic system, and that survivin transcripts are highly expressed in neural progenitor cells, implies that the survivin gene in humans may, similar to vegf, be a modifier in the progression of neural diseases, such as amyotrophic lateral sclerosis, and thus have the potential as a therapeutic target. by regulating cell proliferation and apoptosis, the two zebrafish survivin genes, birc5a and birc5b, play important roles in multiple biologic systems. although we have not examined all organs during development; nor have we yet evaluated its role in the eye; the prominent effects of birc gene depletion on the cardiac, neurovascular and hematopoietic systems in the zebrafish embryo suggest that survivin has temporal and tissue-specific properties. in view of the apparent functional overlap with the murine and human survivin orthologues, the zebrafish model provides an exceptional opportunity to examine the physiologic relevance of the complex molecular and biochemical pathways that govern cell survival and apoptosis. the insights gained will lead to the development of safer, targeted therapeutics for a spectrum of cardio-vascular, hematopoeitic and neurologic diseases, and a better understanding of the etiology and genetics of an array of congenital diseases. 
there are zebrafish survivin genes with overlapping expression patterns during early development, prominently in neural and vascular structures. to gain further insights into the properties of survivin, we used the zebrafish model. overall, with the zebrafish model, we identify survivin as a key regulator of neurogenesis, vasculo-angiogenesis, hematopoiesis and cardiogenesis. these properties of survivin, which are consistent with those identified in mice, indicate that its functions are highly conserved across species, and point to the value of the zebrafish model in understanding the role of this iap in the pathogenesis of human disease, and for exploring its potential as a therapeutic target. survivin is a member of the inhibitor of apoptosis protein family, that uniquely also promotes mitosis and cell proliferation. fully delineating the properties of survivin in vivo in mice has been confounded by early lethal phenotypes following survivin gene inactivation. postnatally, survivin is hardly detected in most tissues, but is upregulated in all cancers, and as such, is a potential therapeutic target. normal growth and development of organisms requires maintenance of a dynamic balance between systems that promote cell survival and those that induce apoptosis. the phenotypes induced by morpholino knockdown of one survivin gene, were rescued by overexpression of the other, indicating that the birc paralogs may compensate for each.
 normal growth and development of organisms requires maintenance of a dynamic balance between systems that promote cell survival and those that induce apoptosis. the molecular mechanisms that regulate these processes remain poorly understood, and thus further in vivo study is required. survivin is a member of the inhibitor of apoptosis protein family, that uniquely also promotes mitosis and cell proliferation. postnatally, survivin is hardly detected in most tissues, but is upregulated in all cancers, and as such, is a potential therapeutic target. prenatally, survivin is also highly expressed in several tissues. fully delineating the properties of survivin in vivo in mice has been confounded by early lethal phenotypes following survivin gene inactivation.  to gain further insights into the properties of survivin, we used the zebrafish model. there are zebrafish survivin genes with overlapping expression patterns during early development, prominently in neural and vascular structures. morpholino-induced depletion of birc5a causes profound neuro-developmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. similar abnormalities, all less severe except for hematopoiesis, were evident with suppression of birc5b. the phenotypes induced by morpholino knockdown of one survivin gene, were rescued by overexpression of the other, indicating that the birc paralogs may compensate for each. the potent vascular endothelial growth factor also entirely rescues the phenotypes induced by depletion of either birc5a and birc5b, highlighting its multi-functional properties, as well as the power of the model in characterizing the activities of growth factors.  overall, with the zebrafish model, we identify survivin as a key regulator of neurogenesis, vasculo-angiogenesis, hematopoiesis and cardiogenesis. these properties of survivin, which are consistent with those identified in mice, indicate that its functions are highly conserved across species, and point to the value of the zebrafish model in understanding the role of this iap in the pathogenesis of human disease, and for exploring its potential as a therapeutic target. 