death executioner Bcl-2 homologue
Bcl-2 family members as regulators of the cell death hierarchy: ced-9 in C. elegans ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central role in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. The CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protects cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduces the cell death induced by ced-3 in Drosophila SL2 cells. Apoptosis of SL2 cells that is induced by a Drosophila cell-death gene, reaper, is partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. SL2 cells provide a unique systems for dissecting the main machinery of cell death (Hisahara, 1998).
Gain-of-function mutations in the Caenorhabditis elegans gene egl-1 cause the HSN neurons to undergo programmed cell death. By contrast, a loss-of-function egl-1 mutation prevents most if not all somatic programmed cell deaths. The egl-1 gene negatively regulates the ced-9 gene, which protects against cell death and is a member of the bcl-2 family. Searches of current nucleotide and protein databases using various BLAST programs have identified no known sequences or proteins with significant similarity to the egl-1 gene or the EGL-1 protein. egl-1 therefore encodes a novel protein of 91 amino acids with no apparent hydrophobic stretches (indicative of transmembrane domains). The EGL-1 protein contains a nine amino acid region similar to the Bcl-2 homology region 3 (BH3) domain but does not contain a BH1, BH2, or BH4 domain, suggesting that EGL-1 may be a member of a family of cell death activators that includes the mammalian proteins Bik, Bid, Harakiri, and Bad. The EGL-1 and CED-9 proteins interact physically. This paper proposes that EGL-1 activates programmed cell death by binding to and directly inhibiting the activity of CED-9, perhaps by releasing the cell death activator CED-4 from a CED-9/CED-4-containing protein complex (Conradt, 1998).
The Caenorhabditis elegans gene ced-9 prevents cells from undergoing programmed cell death and
encodes a protein similar to the mammalian cell-death inhibitor Bcl-2. The CED-9
protein is a substrate for the C. elegans cell-death protease CED-3, which is a member of a family of
cysteine proteases first defined by CED-3 and human interleukin-1beta converting enzyme (ICE). CED-9
can be cleaved by CED-3 at two sites near its amino terminus: the presence of at least one of these
sites is important for complete protection by CED-9 against cell death. Cleavage of CED-9 by CED-3
generates a carboxy-terminal product that resembles Bcl-2 in sequence and in function. Bcl-2 and the
baculovirus protein p35 (which inhibits cell death in different species through a mechanism that depends
on the presence of its cleavage site for the CED-3/ICE family of proteases) inhibit cell death additively in
C. elegans. These results indicate that CED-9 prevents programmed cell death in C. elegans through two
distinct mechanisms: (1) CED-9 may, by analogy with p35, directly inhibit the CED-3 protease by an
interaction involving the CED-3 cleavage sites in CED-9 and (2) CED-9 may directly or indirectly inhibit
CED-3 by means of a protective mechanism similar to that used by mammalian Bcl-2. The second mechanism involves physical interaction with CED-4 in C. elegans and a CED-4 homolog in mammals (Xue, 1997).
Examining the effects of overexpressing cell-death-related genes in
specific C. elegans neurons that normally live, it was demonstrated that
the cell-death genes ced-3, ced-4, and ced-9 all can act cell autonomously to control programmed cell
death. Not only the protective activity of ced-9 but also the killer
activities of ced-3 and ced-4 are likely to be present in cells that normally live. Killing by overexpression of ced-3 does not require endogenous ced-4 function, whereas killing by overexpression of ced-4 is at least in part dependent on endogenous ced-3 function. These results suggest either that ced-4 acts upstream of ced-3 and ced-4 function can be bypassed by high levels of ced-3 activity or that ced-3 and ced-4 act in parallel, with ced-3 perhaps having a greater ability to kill. The finding that ced-4 appears to facilitate the inhibition of ced-3 by ced-9 suggests that ced-9 acts to negatively regulate ced-4. It is proposed that both in
C. elegans and in other organisms a competition between antagonistic protective and killer activities
determines whether specific cells will live or die. These results suggest a genetic pathway for
programmed cell death in C. elegans in which ced-4 acts upstream of or in parallel withced-3, and ced-9
negatively regulates the activity of ced-4 (Shaham, 1996a).
ced-4 encodes two
transcripts; whereas the major transcript can cause programmed cell death, the minor
transcript can act oppositely and prevent programmed cell death, thus defining a novel class of cell
death inhibitors. That ced-4 has both cell-killing and cell-protective functions is consistent with previous
genetic studies. The dual protective and killer functions of the C. elegans
bcl-2-like gene ced-9 are mediated by inhibition of the killer and protective ced-4 functions,
respectively. It is proposed that a balance between opposing ced-4 functions influences the decision of a
cell to live or to die by programmed cell death and that both ced-9 and ced-4 protective functions are
required to prevent programmed cell death (Shaham, 1996b).
Three principal genes are involved in developmental programmed cell death in C. elegans: ced-3 and ced-4 genes are both required for PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes. ced-9 is homologous to the Bcl-2 family, whose role in protecting PCD is illusive; no vertebrate homolog of ced-4 is known. This paper describes the effect of expression of C. elegans ced-4 in yeast. Induction of wild type ced-4 results in rapid focal chromatin condensation and lethality. Mutation of a putative nucleotide binding P-loop motif of CED-4 eliminates the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggests that the phenotype may result from an intrinsic ability of CED-4 to interact with chromatin. Co-expression of ced-9 prevents CED-4-induced chromatin condensation and lethality, and causes the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. It is concluded that CED-4 has a direct role in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. Further studies are required to establish whether the CED-9/CED-4 interaction is required for the activation of CED-3, the Caspase cysteine protease (James, 1997).
Genetic studies suggest that ced-9 controls programmed cell death by regulating
ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease
CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates
that in vivo CED-9 forms a multimeric protein complex with CED-4 and CED-3. Expression of wild-type CED-4 promotes
the ability of CED-3 in mammalian cells to induce apoptosis otherwise inhibited by CED-9. The pro-apoptotic activity of
CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in
their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic
activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results
provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a
molecular basis for the control of programmed cell death in C. elegans (Wu, 1997).
ced-9 is an element of a polycistronic locus that also contains the gene cyt-1, which encodes
a protein similar to cytochrome b560 of complex II of the mitochondrial respiratory chain. ced-9
encodes a 280 amino acid protein showing sequence and structural similarities to the mammalian
proto-oncogene bcl-2. Overexpression of bcl-2 can mimic the protective effect of ced-9 on C. elegans
cell death and can prevent the ectopic cell deaths that occur in ced-9 loss-of-function mutants. These
results suggest that ced-9 and bcl-2 are homologs and that the molecular mechanism of programmed
cell death has been conserved from nematodes to mammals (Hengartner, 1994).
Programmed cell death (PCD) is regulated by multiple evolutionarily conserved
mechanisms to ensure the survival of the cell. This study describes pvl-5,
a gene that likely regulates PCD in
Caenorhabditis elegans. In wild-type hermaphrodites at the L2
stage there are 11 Pn.p hypodermal cells in the ventral
midline arrayed along the anterior-posterior axis and 6 of these
cells become the vulval precursor cells. In pvl-5(ga87)
animals, there are fewer Pn.p cells (average of 7.0) present at
this time. Lineage analysis reveals that the missing Pn.p
cells die around the time of the L1 molt in a manner that often
resembles the programmed cell deaths that occur normally in C.
elegans development. This Pn.p cell death is suppressed by
mutations in the caspase gene ced-3 and in the bcl-2
homolog ced-9, suggesting that the Pn.p cells are dying
by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death
is not suppressed by loss of ced-4 function. ced-4
(Apaf-1) is required for all previously known apoptotic cell deaths
in C. elegans. This suggests that loss of pvl-5
function leads to the activation of a ced-3-dependent,
ced-4-independent form of PCD and that pvl-5 may
normally function to protect cells from inappropriate activation of
the apoptotic pathway (Joshi, 2004).
Genetic analyses in C. elegans have been instrumental in the elucidation of the central cell-death machinery, which is conserved from C. elegans to mammals. One possible difference that has emerged is the role of mitochondria. By releasing cytochrome c, mitochondria are involved in the activation of caspases in mammals. However, there has previously been no evidence that mitochondria are involved in caspase activation in C. elegans. This study shows that mitochondria fragment in cells that normally undergo programmed cell death during C. elegans development. Mitochondrial fragmentation is induced by the BH3-only protein EGL-1 and can be blocked by mutations in the bcl-2-like gene ced-9, indicating that members of the Bcl-2 family might function in the regulation of mitochondrial fragmentation in apoptotic cells. Mitochondrial fragmentation is independent of CED-4/Apaf-1 and CED-3/caspase, indicating that it occurs before or simultaneously with their activation. Furthermore, DRP-1/dynamin-related protein, a key component of the mitochondrial fission machinery, is required and sufficient to induce mitochondrial fragmentation and programmed cell death during C. elegans development. These results assign an important role to mitochondria in the cell-death pathway in C. elegans (Jagasia, 2005).
Temporal control of programmed cell death is necessary to ensure that cells
die at only the right time during animal development. How such temporal
regulation is achieved remains poorly understood. In some Caenorhabditis
elegans somatic cells, transcription of the egl-1/BH3-only gene
promotes cell-specific death. The EGL-1 protein inhibits the CED-9/Bcl-2
protein, resulting in the release of the caspase activator CED-4/Apaf-1.
Subsequent activation of the CED-3 caspase by CED-4 leads to cell death.
Despite the important role of egl-1 transcription in promoting CED-3
activity in cells destined to die, it remains unclear whether the temporal
control of cell death is mediated by egl-1 expression. This study show shows
that egl-1 and ced-9 play only minor roles in the death of
the C. elegans tail-spike cell, demonstrating that temporal control
of tail-spike cell death can be achieved in the absence of egl-1. The timing of the onset of tail-spike cell death is
controlled by transcriptional induction of the ced-3 caspase. The developmental expression pattern of ced-3 has been characterized; in the tail-spike cell, ced-3 expression is induced shortly
before the cell dies, and this induction is sufficient to promote the demise
of the cell. Both ced-3 expression and cell death are dependent on
the transcription factor PAL-1, the C. elegans homolog of the
mammalian tumor suppressor gene Cdx2. PAL-1 can bind to the ced-3
promoter sites that are crucial for tail-spike cell death, suggesting that it
promotes cell death by directly activating ced-3 transcription. These
results highlight a role that has not been described previously for the
transcriptional regulation of caspases in controlling the timing of cell death
onset during animal development (Maurer, 2007).
To obtain insight into the role of the retinoblastoma susceptibility gene
(Rb; also known as Rb1) in apoptosis,
Caenorhabditis elegans mutants lacking a functional lin-35
RB gene were analyzed. The loss of lin-35 function results in a
decrease in constitutive germ cell apoptosis. Evidence is presented that
lin-35 promotes germ cell apoptosis by repressing the expression of
ced-9, an anti-apoptotic C. elegans gene that is orthologous
to the human proto-oncogene BCL2. Furthermore, the genes
dpl-1 DP, efl-1 E2F and efl-2 E2F were also shown to promote
constitutive germ cell apoptosis. However, in contrast to lin-35,
dpl-1 (and probably also efl-1 and efl-2) promotes germ
cell apoptosis by inducing the expression of the pro-apoptotic genes
ced-4 and ced-3, which encode an APAF1-like adaptor protein
and a pro-caspase, respectively. Based on these results, it is proposed that
C. elegans orthologs of components of the RB tumor suppressor complex
have distinct pro-apoptotic functions in the germ line and that the
transcriptional regulation of components of the central apoptosis machinery is
a critical determinant of constitutive germ cell apoptosis in C.
elegans. Finally, lin-35, dpl-1 and
efl-2, but not efl-1, function either downstream of or in
parallel to cep-1 p53 (also known as TP53) and egl-1
BH3-only were shown to cause DNA damage-induced germ cell apoptosis. These results have implications for the general mechanisms through which RB-like proteins control
gene expression, the role of RB-, DP- and E2F-like proteins in apoptosis, and
the regulation of apoptosis (Schertel, 2007).
The developmental control of apoptosis is fundamental and important. The Caenorhabditis elegans Bar homeodomain transcription factor CEH-30 is required for the sexually dimorphic survival of the male-specific CEM (cephalic male) sensory neurons; the homologous cells of hermaphrodites undergo programmed cell death. It is proposed that the cell-type-specific anti-apoptotic gene ceh-30 is transcriptionally repressed by the TRA-1 transcription factor, the terminal regulator of sexual identity in C. elegans, to cause hermaphrodite-specific CEM death. The established mechanism for the regulation of specific programmed cell deaths in C. elegans is the transcriptional control of the BH3-only gene egl-1, which inhibits the Bcl-2 homolog ced-9; similarly, most regulation of vertebrate apoptosis involves the Bcl-2 superfamily. In contrast, ceh-30 acts within the CEM neurons to promote their survival independently of both egl-1 and ced-9. Mammalian ceh-30 homologs can substitute for ceh-30 in C. elegans. Mice lacking the ceh-30 homolog Barhl1 show a progressive loss of sensory neurons and increased sensory-neuron cell death. Based on these observations, it is suggested that the function of Bar homeodomain proteins as cell-type-specific inhibitors of apoptosis is evolutionarily conserved (Schwartz, 2007).
Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. A mammalian analog of this caspase-regulatory system has been identified in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-XL bind and suppress NALP1, reducing caspase-1 activation and interleukin-1β (IL-1β) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1β production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1β production. The findings reveal an interaction of host defense and apoptosis machinery (Bruey, 2007).
Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous
overexpression within this system, expression of the human bcl-2 gene was targeted in mice by using the promoter of the vav gene, which is active throughout this
compartment but rarely outside it. The vav-bcl-2 transgene is expressed in essentially all nucleated cells of hematopoietic tissues but not notably in
nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice display increases in myeloid cells as well as a marked elevation in B and T
lymphocytes. The spleen is enlarged and the lymphoid follicles expanded. Although total thymic cellularity is normal, T cell development is altered: cells at the
very immature and most mature stages are increased, whereas those at the intermediate stage are decreased. Unexpectedly, blood platelets are reduced by
half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remain cytokine
dependent, and the frequency of most progenitor and preprogenitor cells is normal. Macrophage progenitors are less frequent and yield smaller colonies,
however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhances clonogenic
survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the
role of apoptosis in the development and homeostasis of this compartment (Ogilvy, 1999).
Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival
member of the Bcl-2 family, which is widely expressed. To explore its physiological role, the bcl-w gene in the mouse was inactivated by homologous
recombination. Mice that lack Bcl-w are viable, healthy, and normal in appearance. Most tissues exhibit typical histology, and hematopoiesis is unaffected,
presumably due to redundant function with other pro-survival family members. Although female reproductive function is normal, the males are infertile. The testes
developed normally, and the initial, prepubertal wave of spermatogenesis is largely unaffected. The seminiferous tubules of adult males, however, are
disorganized, contained numerous apoptotic cells, and produce no mature sperm. Both Sertoli cells and germ cells of all types are reduced in number, the most
mature germ cells being the most severely depleted. The bcl-w-/- mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some
cases of human male sterility (Print, 1998).
Bcl-x is a member of the Bcl2 family and has been
suggested to be important for the survival and maturation
of various cell types including the erythroid lineage. To
define the consequences of Bcl-x loss in erythroid cells and
other adult tissues, mice conditionally
deficient in the Bcl-x gene were generated using the Cre-loxP
recombination system. The temporal and spatial excision
of the floxed Bcl-x locus was achieved by expressing the Cre
recombinase gene under control of the MMTV-LTR. By the
age of five weeks, Bcl-x conditional mutant mice exhibit
hyperproliferation of megakaryocytes and a decline in the
number of circulating platelets. Three-month-old animals
suffer from severe hemolytic anemia, hyperplasia of
immature erythroid cells and profound enlargement of the
spleen. Bcl-x is only required for the
survival of erythroid cells at the end of maturation, which
includes enucleated reticulocytes in circulation. The
extensive proliferation of immature erythroid cells in the
spleen and bone marrow might be the result of a fast
turnover of late red blood cell precursors and accelerated
erythropoiesis in response to tissue hypoxia. The increase
in cell death of late erythroid cells is independent from the
proapoptotic factor Bax, as demonstrated in conditional
double mutant mice for Bcl-x and Bax. Mice conditionally
deficient in Bcl-x permitted a study of the
effects of Bcl-x deficiency on cell proliferation, maturation
and survival under physiological conditions in an adult
animal (Wagner, 2000).
It is suggested that the function of Bcl-x
as a cell survival factor might not only be restricted to
nucleated cells where classical markers of apoptotic cell death
can be analyzed. Increased numbers of reticulocytes in the
Bcl-x mutants indicate that the bone marrow and spleen
are responding to the anemic situation with accelerated
erythropoiesis and increased release of enucleated
erythrocytes. However, Bcl-x-deficient mice still suffer from
severe anemia, which could suggest that these maturing
erythrocytes have a shorter half-life and hemolyze prematurely.
Similarly, erythroid hyperplasia is more pronounced in
individuals with hemolytic anemia than non-hemolytic anemia.
The expansion of Bcl-x function as a survival factor for cells
without a nucleus would demand a new definition for
apoptosis, which is generally believed to be a phenomenon for
nucleated cells. The hypothesis that Bcl-x is important for the
survival of reticulocytes is supported by earlier findings on in
vitro differentiated mouse and human erythroid cells. The translation of the Bcl-x protein is
sharply increased at the time of maximal hemoglobin synthesis
and remains to accumulate when the majority of erythroblasts
have undergone enucleation to form reticulocytes (Wagner, 2000).
It is known from recent studies that Bcl-x regulates cell
survival by at least two distinct mechanisms: heterodimerization
with other Bcl2 family members and sustained ion-channel
formation. The configuration of ion channels
might be the more potent function in erythroid cells since the
Bcl-x/Bax counteractive mechanism does not appear to regulate
cell survival. These ion channels might control processes such
as mitochondrial ATP/ADP exchange or cytochrome C release.
It can therefore be assumed, that Bcl-x is important until the
reticulocyte stage when mitochondria are still present.
Mitochondria are progressively eliminated from mature
erythrocytes as they meet their energy needs by anaerobic
glycolysis instead of the Krebs cycle. If Bcl-x function is largely
restricted to mitochondria in RBCs it can be predicted that
its role as a survival factor, pore-channel-forming unit or
countermeasure against free radicals has to diminish at the very
end of erythrocyte maturation (Wagner, 2000).
Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. bak-/- mice are developmentally normal and reproductively fit and fail to develop any age-related disorders. However, when Bak-deficient mice are mated to Bax-deficient mice to create mice lacking both genes, the majority of bax-/-bak-/- animals die perinatally with fewer than 10% surviving into adulthood. bax-/-bak-/- mice display multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis (Lindsten, 2000).
A study has been carried out of roles of two anti-apoptotic members
of the Bcl2 family, Bcl-w and Bcl-xL, in regulating the
survival of sensory neurons during development. Microinjection was used to introduce expression plasmids containing
Bcl-w and Bcl-xL cDNAs in the sense and antisense
orientations into the nuclei of BDNF-dependent nodose
neurons and NGF-dependent trigeminal neurons at stages
during and after the period of naturally occurring neuronal
death. While overexpression of either protein promotes
neuronal survival in the absence of neurotrophins and
microinjection of antisense constructs reduce neuronal
survival in the presence of neurotrophins, the magnitude
of these effects changes with age. Whereas Bcl-w
overexpression becomes more effective in promoting
neuronal survival with age, Bcl-xL overexpression becomes
less effective, and whereas antisense Bcl-w becomes much
more effective in killing neurotrophin-supplemented
neurons with age, antisense Bcl-xL becomes much less
effective in killing these neurons. There is a marked
increased in Bcl-w mRNA and Bcl-w immunoreactive
neurons and a decrease in Bcl-xL mRNA and Bcl-xL
immunoreactive neurons in the trigeminal and nodose
ganglia over this period of development. These results
demonstrate that both Bcl-w and Bcl-xL play an important
anti-apoptotic role in regulating the survival of NGF- and
BDNF-dependent neurons, and that reciprocal changes
occur in the relative importance of these proteins with age.
Whereas Bcl-xL plays a more important role during the
period of naturally occurring neuronal death, Bcl-w plays
a more important role at later stages (Middleton, 2001).
Male mice deficient in BCLW, a death-protecting member of the BCL2 family, are sterile due to an arrest in spermatogenesis that is associated with a gradual loss of germ cells and Sertoli cells from the testis. Since Bclw is expressed in both Sertoli cells and diploid male germ cells, it has been unclear which of these cell types requires BCLW in a cell-autonomous manner for survival. To determine whether death of Sertoli cells in Bclw mutants is influenced by the protracted loss of germ cells, testes from Bclw/c-kit double mutant mice, which lack germ cells from birth, were examined. Loss of BCLW-deficient Sertoli cells occurs in the absence of germ cells, indicating that germ cell death is not required to mediate loss of Sertoli cells in BCLW-deficient mice. This suggests that Sertoli cells require BCLW in a cell-intrinsic manner for long-term survival. The loss of Sertoli cells in Bclw mutants commences shortly after Sertoli cells have become postmitotic. In situ hybridization analysis indicates that Bclw is expressed in Sertoli cells both before and after exit from mitosis. Therefore, Bclw-independent pathways promote the survival of undifferentiated, mitotic Sertoli cells. BAX and BAK, two closely related death-promoting members of the BCL2 family, are expressed in Sertoli cells. To determine whether either BAX or BAK activity is required for Sertoli cell death in Bclw mutant animals, survival of Sertoli cells was analyzed in Bclw/Bax and Bclw/Bak double homozygous mutant mice. While mutation of Bak has no effect, ablation of Bax suppresses the loss of Sertoli cells in Bclw mutants. Thus, BCLW mediates survival of postmitotic Sertoli cells in the mouse by suppressing the death-promoting activity of BAX (Ross, 2001).
In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. The pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, Bax is shown to be downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages (Stallock, 2003).
A significantly increased number of ectopic germ cells is present in Bax-/- embryos. The ectopic germ cells are developmentally delayed. They retain expression of early PGC markers and retain motility. This shows that signals from the gonad regulate expression of PGC markers and inhibit their motility. Bax-/- ectopic germ cells occupy many positions in the embryo. However, they do not grow, and their numbers dwindle until by E18.5 very few can be found. Since these mice have not been reported to have an increased incidence of germline tumors, the data suggest that mice have a back-up mechanism for removing embryonic migratory germ cells in ectopic locations. Inactivation of Bax protects germ cells against rapid cell death in culture, and against removal of the Steel/Kit signaling interaction in culture. This shows that Bax is downstream of the Kit receptor. However, protection against cell death in culture is a short-term effect, showing that other apoptotic pathways exist in germ cells (Stallock, 2003).
During inner ear development, programmed cell death occurs in specific areas of the otic epithelium but the significance of this death and the molecules involved have remained unclear. An analysis was undertaken of mouse mutants in which genes encoding apoptosis-associated molecules have been inactivated. Disruption of the Apaf1 gene leads to a dramatic decrease in apoptosis in the inner ear epithelium, severe morphogenetic defects and a significant
size reduction of the membranous labyrinth, demonstrating that an
Apaf1-dependent apoptotic pathway is necessary for normal inner ear
development. This pathway most probably operates through the apoptosome
complex because caspase 9 mutant mice suffer similar defects. Inactivation
of the Bcl2-like (Bcl2l) gene leads to an overall increase in the
number of cells undergoing apoptosis but does not cause any major morphogenetic defects. In contrast, decreased apoptosis is observed in specific locations that suffer from developmental deficits, indicating that proapoptotic isoform(s) produced from Bcl2l might have roles in inner ear development. In Apaf1-/-/Bcl2l-/- double mutant embryos, no cell death could be detected in the otic epithelium, demonstrating that the cell death regulated by the anti-apoptotic Bcl2l isoform (Bcl-XL) in the otic epithelium is Apaf1-dependent. Furthermore, the otic vesicle fails to close completely in all double mutant embryos analyzed. These results indicate important roles for both Apaf1 and Bcl2l in inner ear development (Cecconi, 2004).
Bcl-2 is a death repressor that protects cells from apoptosis mediated by a variety of stimuli. Bcl-2 expression is regulated by both pro- and anti-angiogenic factors; thus, it may play a central role during angiogenesis. However, the role of bcl-2 in vascular development and growth of new vessels requires further delineation. In this study, the physiological role of bcl-2 was investigated in development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Mice deficient in bcl-2 exhibit a significant decrease in retinal vascular density compared to wild-type mice. This was attributed to a decreased number of endothelial cells and pericytes in retinas from bcl-2-/- mice. In bcl-2-/- mice, delayed development of retinal vasculature and remodeling was observed, and a significant decrease in the number of major arteries, which branch off from near the optic nerve. Interestingly, hyaloid vessel regression, an apoptosis-dependent process, was not affected in the absence of bcl-2. The retinal vasculature of bcl-2-/- mice exhibits a similar sensitivity to hyperoxia-mediated vessel obliteration compared to wild-type mice during OIR. However, the degree of ischemia-induced retinal neovascularization is significantly reduced in bcl-2-/- mice. These results suggest that expression of bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during OIR (Wang, 2005).
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