ESPE Abstracts

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.