ESPE Abstracts (2014) 82 FC4.2

The Effect of grB10-Deficiency in Zebrafish: A Translational Animal Model to Study Human Growth

Chiara De Leonibusa, Irene Barinaga-Rementeria Ramirezb, Adam Hurlstoneb, Peter Claytona & Adam Stevensa

aManchester Academic Health Sciences Centre, Royal Manchester Children’s Hospital, Manchester, UK; bFaculty of Life Sciences, The Michael Smith Building, University of Manchester, Manchester, UK

Background: GRB10 negatively regulates IGF1 signalling, influences growth and promoter polymorphisms are associated with GH response1. GRB10 knockout-mouse models display foetal overgrowth2, however, the mouse model has only partial similarity to human growth3. There is evidence that the zebrafish is an appropriate model to study growth4 and has the advantage of being easily genetically manipulated.

Objective: To use zebrafish as a vertebrate animal model to study the effects of GRB10-deficiency on growth and development.

Methods: GRB10 expression was analysed by developmental stages and tissue distribution using qPCR. Transgenic zebrafish were obtained by injecting a splice-blocking morpholino (MO) into WT embryos to generate GRB10-deficiency. Dose-dependent titration of MO injection was performed (0.1, 0.5, 0.7, and 1 mM) into one-cell stage embryos with mock-injected zebrafish (dye only) as a control. Growth measurements and embryo development were assessed by time-lapse microscopy from birth up to 72 h post-fertilization.

Results: GRB10 gene expression changes were identified during the growth phases, with a major peak in early embryogenesis followed by a gradual age-dependent decrease until the adult stage. GRB10 was widely expressed in all adult tissues examined, with the highest expression in kidney, brain, ovaries, and bone. We observed a dose-dependent overgrowth in GRB10-deficient embryos compared to controls, with proportional increase in length, head size, and eye distance (P<0.05), along with gradual increase in mortality compared to controls (22, 33, 43, and 46% at MO increasing dose vs 8%, P=0.013). Time-lapse microscopy showed GRB10-deficient embryos progressing through the first 72 h of embryogenesis at the same rate as controls, with no significant change in pigmentation and organogenesis.

Conclusion: Our results indicate a major role of GRB10 in zebrafish development. These findings support zebrafish as a vertebrate model whereby genetic dysregulation of GRB10 uleads to proportionate overgrowth, implying important effects on growth suppression and whole-body size.

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