Endocrine Abstracts

Introduction: Patients with aryl hydrocarbon receptor-interacting protein (AIP) gene mutations are predisposed to large, invasive, GH- or PRL-secreting pituitary tumours, occurring at a younger age and poorly responsive to treatment. The zebrafish (ZF) model provides anatomical and functional similarities to human neuroendocrine system.

Methods: AIP knock down (KD) ZF embryos were generated using antisense morpholino oligonucleotides injected at one-cell stage. Control embryos were injected with 5-base mispaired oligonucleotide as control morpholinos (CM). Wild type (WT) embryos from the same batch served as uninjected controls. All embryos were incubated in the same conditions for 5 days, and observed during development. At 120 hours post fertilization (hpf) whole mount immunostaining of all embryos was performed with anti-PRL antibodies (rabbit anti-salmon polyclonal 1:2000). A total of 15 embryos (5 from each group) were randomly selected for digital microscopy. Pituitary staining was assessed by image analysis software (NIH ImageJ 1.48v).

Results: Overall developmental delay and retardation was observed in the AIP KD compared to WT and CM control groups. KD embryos exhibited reduced total body length, transitory midbrain enlargement, pericardium enlargement and swim bladder under-development. Assessed by the PRL staining, pituitary in the AIP morphants appeared to be larger, ventrally shifted and round shaped compared to oval or kidney shaped pituitary in the WT. Pituitary size in AIP morphants (1621.9±87.2 μm²) was significantly larger then in WT (574.1±357.8 μm² P=0.04) and CM (626.0±223.6 μm² P=0.02) with no statistical difference between the two control groups (P=0.90).

Conclusion: AIP Morpholino Knock Down zebrafish embryos demonstrate brain, pericardium, and swim bladder anomalies and general developmental delay, pointing to wide developmental role of AIP gene. AIP morphant embryos exhibit larger surface of PRL immunostaining in the pituitary compared to controls suggesting possible increase in proliferative activity (hyperplasia or tumour) at pituitary level in the absence of AIP gene function.