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Biology subjects

Kowald, S.

Publications and source records attributed to Kowald, S..

2 recordsLinked to original sources

Automated microinjection for zebrafish xenograft models

Zebrafish xenograft models have been increasingly recognized for their ability to predict patient responses to cancer therapeutics, suggesting their potential as diagnostic tools in clinical settings. However, these models require the precise microinjection of cancer cell suspensions in many small and fragile zebrafish larvae. Manual injections are so challenging that, even after months of training, variability in experimental results persists among researchers. This limits the uptake and deployment of zebrafish xenograft models for clinical use and drug discovery. To address this challenge, we have designed, built, and validated an automated microinjection robot. Combined results of injections into the vasculature, perivitelline space, and hindbrain ventricle demonstrated an average injection success rate of approximately 60%, with a larvae survival rate exceeding 70%, comparable to manual injections using a traditional micromanipulator. Notably, the full automated mode was twice as fast as manual injections. This automation of the microinjection process significantly reduces the need for extensive personnel training while it enhances reproducibility, efficiency, and accuracy, paving the way for more extensive use of zebrafish xenograft models in drug discovery and patient diagnostics.

cancer biology↗

FoxO transcription factors actuate the formative pluripotency specific gene expression programme

Naive pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naive pluripotency-specific transcription factors (TFs). Upon exiting naive pluripotency, ES cells transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage-choice. However, the mechanisms underlying disengagement from the naive GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localization of FoxO TFs in naive ESCs. PTEN-mediated reduction of AKT activity upon exit from naive pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for transition from the naive to the formative pluripotent state. Our work uncovers a pivotal role for FoxO TFs and AKT signalling in mechanisms establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition.

developmental biology↗