In vitro studies have suggested that the Cables1 gene regulates epithelial cell proliferation, whereas other studies suggest a role in promoting neural differentiation. In efforts to clarify the functions of Cables1 in vivo, we conducted gain- and loss-of-function studies targeting its ortholog (cables1) in the zebrafish embryo. Similar to rodents, zebrafish cables1 mRNA expression is detected most robustly in embryonic neural tissues. Antisense knockdown of cables1 leads to increased numbers of apoptotic cells, particularly in brain tissue, in addition to a distinct behavioral phenotype, characterized by hyperactivity in response to stimulation. Apoptosis and the behavioral abnormality could be rescued by co-expression of a morpholino-resistant cables1 construct. Suppression of p53 expression in cables1 morphants partially rescued both apoptosis and the behavioral phenotype, suggesting that the phenotype of cables1 morphants is due in part to p53-dependent apoptosis. Alterations in the expression patterns of several neural transcription factors were observed in cables1 morphants during early neurulation, suggesting that cables1 is required for early neural differentiation. Ectopic overexpression of cables1 strongly disrupted embryonic morphogenesis, while overexpression of a cables1 mutant lacking the C-terminal cyclin box had little effect, suggesting functional importance of the cyclin box. Lastly, marked reductions in p35, but not Cdk5, were observed in cables1 morphants. Collectively, these data suggest that cables1 is important for neural differentiation during embryogenesis, in a mechanism that likely involves interactions with the Cdk5/p35 kinase pathway.
The CDK5 and ABL1 Enzyme Substrate 1 (Cables1/ik3-1) gene, a member of the cyclin superfamily, was identified in two independent screens for genes that interact with the cyclin-dependent kinase (Cdk) family of serine/threonine protein kinases (Matsuoka et al., 2000; Zukerberg et al., 2000). In a yeast-two hybrid screening assay, Cables1 protein interacted strongly with Cdk5 (a non-cell cycle-associated kinase required for neural differentiation), while weaker interactions were also observed with cell cycle-associated kinases Cdk2 and Cdk3. Subsequent in vitro studies utilizing COS7 cells, E15 mouse brain lysates and cultured primary rat cortical neurons suggested that Cables1 can function in two distinct cellular contexts: Firstly, Cables1 promoted protein–protein interactions among c-ABL kinase, Cdk5 and p35 that enhanced tyrosine phosphorylation of Cdk5 (Zukerberg et al., 2000); secondly, Cables1 could negatively regulate cell proliferation, by augmenting inhibitory phosphorylation cascades that suppress cyclin-dependent kinase activity (Wu et al., 2001). While informative, these in vitro studies have yet to provide definitive insights into the functions of the Cables1 gene in vivo, particularly during early development.
Although Cables1 knockout mice are viable, early (non-congenic) strains were notably subfertile, with evidence of fetal resorption in pregnant dams. These observations suggest that embryonic development is compromised in Cables1 knockouts. Interpretation of these data remains difficult, however, due to the potential redundancy of a related gene (Cables2) in mice. In light of multiple studies reporting a loss of Cables1 expression in selected tumor types there remains a strong rationale to clarify the in vivo functions of the Cables1 gene.
To broaden our understanding of Cables1 functions in vivo, we have performed gain- and loss-of-function studies of the zebrafish Cables1 ortholog (cables1; NM_ 001105665.1). Importantly, the lack of a functional paralog in this species permitted a direct investigation of cables1 functions in vivo. The data from these studies support the notion that cables1 is critically important for neural development in the zebrafish embryo. The developmental impact of cables1 suppression appears substantially more dramatic than that observed in murine or avian models. In the zebrafish, knockdown of cables1 results in disruptions in the expression of multiple neural transcription factors, increased apoptosis in embryonic neural tissues, and subsequent behavioral defects characterized by hyperactivity in response to stimulation. In addition, we present evidence suggesting that the embryonic functions of cables1 likely involve interactions with the Cdk5/p35 kinase pathway. Collectively, these data from the zebrafish model provide in vivo evidence supporting an essential functional role for cables1 during embryonic neural differentiation.
|Storage||Storage: store at 2-8C; Stability: The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.|
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|Research Areas||Cancer Research|
Brain Necrosis in cables1 Morphants
Embryos microinjected at the one-cell stage with a control morpholino oligonucleotide (MO) were indistinguishable from wild-type (non-injected) embryos. By contrast, microinjecting a morpholino oligonucleotide targeting zebrafish cables 1 (cabMO; 2 ng/embryo) led to visible developmental defects, most notably affecting anterior (neural) tissues. By 24 hpf, the most readily observable phenotype was the appearance of cloudy (necrotic) tissue in midbrain and hindbrain ; in a representative experiment, this phenotype was observed in 81% (58/72) of morphants, in a pattern that was highly consistent among injected embryos.
To ensure target specificity of cabMO, rescue experiments were performed using a MO-resistant mRNA construct (cabRESC) encoding the complete open reading frame of cables1. Co-injection of cabRESC (15 pg/embryo) into cabMO-injected embryos markedly attenuated the brain phenotype; substantially fewer embryos (25/78, 32%) showed evidence of brain necrosis, while the proportion of embryos resembling control-injected embryos more than doubled (29/78; 37%, vs. 17% for cabMO-injected embryos). The remaining embryos showed mild overexpression phenotypes (described below).
Increased Apoptosis in cables1 Morphants
It was previously reported that cables1 can modulate apoptosis, via both p53-dependent and p53-independent pathways (Tsuji et a., 2002). To determine whether or not the necrotic appearance of brain tissues in cables1 morphants resulted from increased apoptosis, embryos were assayed for DNA fragmentation at 18 hpf using the terminal dUTP nick-end labeling (TUNEL) assay. Relative to control-injected embryos, a significant increase was observed in the mean number of TUNEL-positive cells (TUNEL index, see Materials and Methods Section) in cables1 morphants (Fig. 3B; P <0.001), with the highest intensity of TUNEL-positive cells observed in regions corresponding to areas of necrotic degeneration.
We next examined whether or not the increased apoptosis could be rescued by co-expressing cabRESC. As shown, the number of TUNEL-positive cells in cabMO-injected embryos co-injected with cabRESC was significantly reduced relative to embryos injected with cabMO alone (P <0.001). The TUNEL index of cabRESC-injected embryos also did not differ significantly from the mean TUNEL index in control-injected embryos (P >0.05), indicating a robust rescue of the phenotype.
To determine if the increased number of TUNEL-positive cells in cabMO-injected embryos was mediated by a p53-dependent apoptotic pathway, endogenous p53 expression was suppressed in cabMO-injected embryos by co-injection with a MO targeting p53 (p53MO). Co-injection of p53MO markedly rescued apoptosis in cabMO-injected embryos, with significantly fewer TUNEL-positive cells detected relative to embryos injected with cabMO alone (P <0.01). The mean TUNEL index of p53MO co-injected embryos was similar to the mean TUNEL index of cables1 morphants co-injected with cabRESC (P >0.05), but was still significantly greater (P <0.05) than the mean TUNEL index of control-injected embryos. These data indicate a significant, but incomplete, rescue of apoptosis by suppressing p53 expression.