ESPE Abstracts (2015) 84 P-2-401

GRB10 Knockdown in Zebrafish is Associated with Decreased Weight-to-length Ratio without Alterations in AKT and ERK Activity: A Model to Study Human Growth Regulation

Chiara De Leonibusa, Jack Broadbentb, Philip Murraya, Joseph Whiteheadb, Adam Hurlstoneb, Holly Shielsb, Peter Claytona & Adam Stevensa

aFaculty of Medical and Human Sciences, Institute of Human Development, University of Manchester and Manchester Academic Health Science Centre, Royal Manchester Children’s Hospital, Manchester, UK; bFaculty of Life Sciences, University of Manchester, Manchester, UK

Background: In humans GRB10 negatively regulates GH and IGF1 signaling predominantly via altering phosphorylation of PI3K/mTOR/AKT and MEK/ERK pathways which relate to both growth and metabolic function. We have previously shown that Grb10 knockdown in Zebrafish results in overgrowth with an increase in length and head size. However the impact on weight in relation to length has not been assessed.

Objective: To develop a model to study weight-to-length ratio in Zebrafish and to examine the mechanisms through which Grb10 knockdown mediates overgrowth.

Methods: Grb10 knockdown was obtained by injecting splice-blocking morpholino (MO) into one-cell stage embryos alongside control-injected (CT) Zebrafish. Weight-to-length ratio (mg/mm2) was assessed at 54, 72 and 120 h post-fertilization (hpf); these developmental periods were chosen to model early through late childhood growth. Chemical inhibition of the PI3K/mTOR/AKT (NVPBEZ235) and the MEK/ERK pathways (PD184352) was performed from 30 to 72 hpf. Total and phosphorylated AKT and ERK were evaluated on Western-Blot to assess the activity of phosphorylation on these molecules with and without pathway inhibition.

Results: In the MO Zebrafish (Grb10 knockdown), the weight-to-length ratio significantly decreased over time and the reduction was most significant at 120 hpf (n=8, P<0.001). At all times, weight-to-length ratio was significantly increased in CT vs MO (P<0.05). The comparison by Western-Blot with and without pathway inhibition in MO vs CT samples indicated that global AKT and ERK activities were not affected by Grb10 knockdown.

Conclusions: We now demonstrate in Zebrafish Grb10 knockdown that not only is length increased but weight-to-length ratio is decreased, implying a ‘leaner’ phenotype. However this is not related to the PI3K/mTOR/AKT and MEK/ERK pathways. We have developed a method to study both length and weight changes in genetically modified Zebrafish which may be useful in modelling human disorders of growth and metabolism.