Functional characterization of the Bcl-2 gene family in the zebrafish.

Members of the Bcl-2 protein family control the intrinsic apoptosis pathway. To evaluate the importance of this family in vertebrate development, we investigated it in the zebrafish (Danio rerio). We found that the zebrafish genome encodes structural and functional homologs of most mammalian Bcl-2 family members, including multi-Bcl-2-homology (BH) domain proteins and BH3-only proteins. Apoptosis induction by gamma-irradiation required zBax1 and zPuma, and could be prevented by overexpression of homologs of prosurvival Bcl-2 family members. Surprisingly, zebrafish Bax2 (zBax2) was homologous to mammalian Bax by sequence and synteny, yet demonstrated functional conservation with human Bak. Morpholino knockdown of both zMcl-1a and zMcl-1b revealed their critical role in early embryonic zebrafish development, and in the modulation of apoptosis activation through the extrinsic pathway. These data indicate substantial functional similarity between zebrafish and mammalian Bcl-2 family members, and establish the zebrafish as a relevant model for studying the intrinsic apoptosis pathway.

Delineation of the cell-extrinsic apoptosis pathway in the zebrafish.

The mammalian extrinsic apoptosis pathway is triggered by Fas ligand (FasL) and Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL). Ligand binding to cognate receptors activates initiator caspases directly in a death-inducing signaling complex. In Drosophila, TNF ligand binding activates initiator caspases indirectly, through JNK. We characterized the extrinsic pathway in zebrafish to determine how it operates in a nonmammalian vertebrate. We identified homologs of FasL and Apo2L/TRAIL, their receptors, and other components of the cell death machinery. Studies with three Apo2L/TRAIL homologs demonstrated that they bind the receptors zHDR (previously linked to hematopoiesis) and ovarian TNFR (zOTR). Ectopic expression of these ligands during embryogenesis induced apoptosis in erythroblasts and notochord cells. Inhibition of zHDR, zOTR, the adaptor zFADD, or caspase-8-like proteases blocked ligand-induced apoptosis, as did antiapoptotic Bcl-2 family members. Thus, the extrinsic apoptosis pathway in zebrafish closely resembles its mammalian counterpart and cooperates with the intrinsic pathway to trigger tissue-specific apoptosis during embryogenesis in response to ectopic Apo2L/TRAIL expression.

Human disease-causing NOG missense mutations: effects on noggin secretion, dimer formation, and bone morphogenetic protein binding.

Secreted noggin protein regulates bone morphogenetic protein activity during development. In mice, a complete loss of noggin protein leads to multiple malformations including joint fusion, whereas mice heterozygous for Nog loss-of-function mutations are normal. In humans, heterozygous NOG missense mutations have been found in patients with two autosomal dominant disorders of joint development, multiple synostosis syndrome (SYNS1) and a milder disorder proximal symphalangism (SYM1). This study investigated the effect of one SYNS1 and two SYM1 disease-causing missense mutations on the structure and function of noggin. The SYNS1 mutation abolished, and the SYM1 mutations reduced, the secretion of functional noggin dimers in transiently transfected COS-7 cells. Coexpression of mutant noggin with wild-type noggin, to resemble the heterozygous state, did not interfere with wild-type noggin secretion. These data indicate that the human disease-causing mutations are hypomorphic alleles that reduce secretion of functional dimeric noggin. Therefore, we conclude that noggin has both species-specific and joint-specific dosage-dependent roles during joint formation. Surprisingly, in contrast to the COS-7 cell studies, the SYNS1 mutant was able to form dimers in Xenopus laevis oocytes. This finding indicates that there also exist species-specific differences in the ability to process mutant noggin polypeptides.

Xenopus Dan, a member of the Dan gene family of BMP antagonists, is expressed in derivatives of the cranial and trunk neural crest.

Dan is the founding member of the Dan family of secreted cytokines. All members of this family--which includes Gremlin, Cerberus, Dante, PRDC, and several genes identified as expressed sequence tags in the mouse--characterized to date have been shown to antagonize signaling by the bone morphogenetic protein (BMP) family. During mouse embryogenesis, Dan is expressed in a restricted and dynamic pattern. Major sites of transcription include the somites, the myotome and the cranial, and facial and limb mesenchyme. Xenopus Dan (XDan) shares over 76% amino acid identity with mouse Dan (mDan). Here we report that in Xenopus embryos, XDan is expressed both as a maternal transcript and at later stages in populations of cells associated with the cranial and trunk neural crest. The conservation of Dan expression in cells of the head mesenchyme between Xenopus and mouse embryos suggests an important role for BMP antagonists in these tissues.

In Xenopus embryos, BMP heterodimers are not required for mesoderm induction, but BMP activity is necessary for dorsal/ventral patterning.

The activity of bone morphogenetic protein (BMP) heterodimers has been shown to be more potent than that of homodimers in a number of contexts, including mesoderm induction. Although BMP-2/7 and -4/7 heterodimers are potent inducers of ventral mesoderm in ectodermal explants, we show that they are not a necessary component of the primary mesoderm-inducing signal in intact Xenopus embryos. The secreted BMP antagonists noggin and gremlin both efficiently block mesoderm induction by BMP homo- and heterodimers in animal caps. When these antagonists are ectopically expressed in the ventral marginal zone of early embryos the initial formation of mesoderm as indicated by panmesodermal markers remains unaffected. Only the subsequent dorsal/ventral patterning of this mesoderm appears to be altered, with expression of a number of organizer-specific transcripts observed in the marginal zone where BMP signaling has been abolished. Thus, we conclude that BMPs do not contribute an essential signal to mesodermal induction or patterning until gastrulation. The activities of noggin and gremlin are strikingly different from that of the multifunctional antagonist cerberus, which completely abolishes mesoderm induction when misexpressed during early development.

The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.

Using a Xenopus expression-cloning screen, we have isolated Gremlin, a novel antagonist of bone morphogenetic protein (BMP) signaling that is expressed in the neural crest. Gremlin belongs to a novel gene family that includes the head-inducing factor Cerberus and the tumor suppressor DAN. We show that all family members are secreted proteins and that they act as BMP antagonists in embryonic explants. We also provide support for the model that Gremlin, Cerberus, and DAN block BMP signaling by binding BMPs, preventing them from interacting with their receptors. In addition, Cerberus alone blocks signaling by Activin- and Nodal-like members of the TGF beta superfamily. Therefore, we propose that Gremlin, Cerberus, and DAN control diverse processes in growth and development by selectively antagonizing the activities of different subsets of the TGF beta ligands.

Age-associated mitochondrial DNA deletions in mouse skeletal muscle: comparison of different regions of the mitochondrial genome.

The abundance of mitochondrial DNA (mtDNA) deletions has been shown to increase with age in a number of species and may contribute to the aging process. Estimating the total mtDNA deletion load of an individual is essential in evaluating the potential physiological impact. In this study, we compared three 5-kb regions of the mitochondrial genome: one in the major arc, one in the minor arc, and a third containing the light strand origin of replication. Through PCR analysis of mouse skeletal muscle, we have determined that not all regions produce equal numbers of age-associated deletions. There are, on average, twofold more detectable deletions in the major arc region than in the minor arc region. Deletions that result in the loss of the light strand origin of replication are rarely detected. Furthermore, the mechanism of deletion formation seems to be similar in both the major and minor arcs, with direct repeats playing an important, although not essential, role.