Limits...
Effects of Cyclin Dependent Kinase 9 inhibition on zebrafish larvae

View Article: PubMed Central - PubMed

ABSTRACT

CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as anti-cancer drugs. The zebrafish embryo provides an ideal model to explore the effects of CDK9 inhibition in-vivo. This has not been adequately explored previously at the level of a whole organism. We have compared and contrasted the effects of pharmacological and molecular inhibition of CDK9 on somatic growth, apoptosis and cellular proliferation in zebrafish larvae between 0 to 120 hours post fertilisation (hpf) using flavopiridol, a selective CDK9 antagonist, and CDK9-targeting morpholino. We demonstrate that the inhibition of CDK9 diminishes cellular proliferation and increases apoptosis. Subsequently, it affects somatic growth and development of a number of key embryonic structures including the brain, heart, eye and blood vessels. For the first time, we have localized CDK9 at a subcellular level in whole-mounted larvae.

This works shows, at a high-throughput level, that CDK9 clearly plays a fundamental role in early cellular growth and proliferation.

No MeSH data available.


Related in: MedlinePlus

Kaplan-Meier survival curve following exposure to flavopiridol or morpholino injection. Survival rate in zebrafish embryos following continuous exposure to flavopiridol (at least n = 100 per group) in the range 1–5µM (A), from 24hpf up to 120 hpf or injection with morpholino 0.2ng/embryo (at least n = 100 per group) (B). Surviving embryos were counted every 24 hours until 120hpf.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5134698&req=5

f0001: Kaplan-Meier survival curve following exposure to flavopiridol or morpholino injection. Survival rate in zebrafish embryos following continuous exposure to flavopiridol (at least n = 100 per group) in the range 1–5µM (A), from 24hpf up to 120 hpf or injection with morpholino 0.2ng/embryo (at least n = 100 per group) (B). Surviving embryos were counted every 24 hours until 120hpf.

Mentions: Absence of swim activity, heart beat and tail blood flow were used as criteria to differentiate a viable from a non-viable larva. Kaplan-Meier curve showed that at 120 hours post-fertilisation (hpf), i.e. 96 hour post-exposure, the recorded survival was 92%, 75% and 57%, respectively in the group of larvae exposed to flavopiridol at 1 µM, 3 µM and 5µM (Fig. 1A). In comparison, CDK9 morpholino splice blocking injected embryo group showed 72% survival (Fig. 1B). Embryo phenotypic traits were analyzed at 72 hpf (Fig. 2). At the concentration of 5µM larvae malformations, such as curved body (50%) and edema (82% for both mild and severe) were commonly observed (Fig. 2A and B), whereas at the concentration of 3µM these malformation were less frequent, respectively 17% and 47%, although CDK9 activity was still reduced (Fig. 2C). At 5µM, 40% of embryos were still chorionated and 27% showed reduced total body length, whereas at 3µM these values were 12% and 30%, respectively, compared to control. A dose-dependent inhibition of CDK9 activity was observed at each of the 3 concentrations tested (1, 3 and 5 µM) as a progressive reduction in phosporylation of the target of CDK9, i.e., serine 2 residue of the carboxy-terminal domain (P-Ser2-CTD) in the RNA pol II (Fig. 2C). On the basis of the findings in this dose response studies, flavopiridol 3µM was adopted thereafter in for all subsequent experiments. CDK9 morpholino injected embryos showed similar phenotypic traits as embryos treated with flavopiridol 3µM (Fig. 2B). Once again a range of concentrations of morpholino were tested and a final concentration was selected based on a balance of effective reduction in CDK9 levels, minimal phenotypic abnormalities in whole embryos and embryo survival of greater than 70% at 72hpf.Figure 1.


Effects of Cyclin Dependent Kinase 9 inhibition on zebrafish larvae
Kaplan-Meier survival curve following exposure to flavopiridol or morpholino injection. Survival rate in zebrafish embryos following continuous exposure to flavopiridol (at least n = 100 per group) in the range 1–5µM (A), from 24hpf up to 120 hpf or injection with morpholino 0.2ng/embryo (at least n = 100 per group) (B). Surviving embryos were counted every 24 hours until 120hpf.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134698&req=5

f0001: Kaplan-Meier survival curve following exposure to flavopiridol or morpholino injection. Survival rate in zebrafish embryos following continuous exposure to flavopiridol (at least n = 100 per group) in the range 1–5µM (A), from 24hpf up to 120 hpf or injection with morpholino 0.2ng/embryo (at least n = 100 per group) (B). Surviving embryos were counted every 24 hours until 120hpf.
Mentions: Absence of swim activity, heart beat and tail blood flow were used as criteria to differentiate a viable from a non-viable larva. Kaplan-Meier curve showed that at 120 hours post-fertilisation (hpf), i.e. 96 hour post-exposure, the recorded survival was 92%, 75% and 57%, respectively in the group of larvae exposed to flavopiridol at 1 µM, 3 µM and 5µM (Fig. 1A). In comparison, CDK9 morpholino splice blocking injected embryo group showed 72% survival (Fig. 1B). Embryo phenotypic traits were analyzed at 72 hpf (Fig. 2). At the concentration of 5µM larvae malformations, such as curved body (50%) and edema (82% for both mild and severe) were commonly observed (Fig. 2A and B), whereas at the concentration of 3µM these malformation were less frequent, respectively 17% and 47%, although CDK9 activity was still reduced (Fig. 2C). At 5µM, 40% of embryos were still chorionated and 27% showed reduced total body length, whereas at 3µM these values were 12% and 30%, respectively, compared to control. A dose-dependent inhibition of CDK9 activity was observed at each of the 3 concentrations tested (1, 3 and 5 µM) as a progressive reduction in phosporylation of the target of CDK9, i.e., serine 2 residue of the carboxy-terminal domain (P-Ser2-CTD) in the RNA pol II (Fig. 2C). On the basis of the findings in this dose response studies, flavopiridol 3µM was adopted thereafter in for all subsequent experiments. CDK9 morpholino injected embryos showed similar phenotypic traits as embryos treated with flavopiridol 3µM (Fig. 2B). Once again a range of concentrations of morpholino were tested and a final concentration was selected based on a balance of effective reduction in CDK9 levels, minimal phenotypic abnormalities in whole embryos and embryo survival of greater than 70% at 72hpf.Figure 1.

View Article: PubMed Central - PubMed

ABSTRACT

CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as anti-cancer drugs. The zebrafish embryo provides an ideal model to explore the effects of CDK9 inhibition in-vivo. This has not been adequately explored previously at the level of a whole organism. We have compared and contrasted the effects of pharmacological and molecular inhibition of CDK9 on somatic growth, apoptosis and cellular proliferation in zebrafish larvae between 0 to 120 hours post fertilisation (hpf) using flavopiridol, a selective CDK9 antagonist, and CDK9-targeting morpholino. We demonstrate that the inhibition of CDK9 diminishes cellular proliferation and increases apoptosis. Subsequently, it affects somatic growth and development of a number of key embryonic structures including the brain, heart, eye and blood vessels. For the first time, we have localized CDK9 at a subcellular level in whole-mounted larvae.

This works shows, at a high-throughput level, that CDK9 clearly plays a fundamental role in early cellular growth and proliferation.

No MeSH data available.


Related in: MedlinePlus