Endocrine Abstracts

Background: Aryl hydrocarbon receptor-interacting protein (AIP) has been identified as a tumour suppressor gene in pituitary gland, causing 10% of all familial isolated pituitary adenoma. Patients with heterozygous loss-of-function germline mutation of AIP develop young-onset growth hormone and/or prolactin-secreting pituitary tumours. Homozygous loss of AIP leads to embryonic lethality in several animal models (mouse, fruit fly, round worm). Mouse embryos show cardiac developmental abnormalities.

Objective: The aim of the current study was to generate a zebrafish loss-of-function model for aip in which to explore the cell biological processes affected by heterozygous and homozygous loss of this gene.

Methods: The EMBOSS Needle was used to identify the zebrafish homologue of the human AIP. We used CRISPR/Cas9 gene editing to generate a line of fish carrying loss of function mutations targeting exon 2 in the zebrafish aip gene. Aip RNA expression was examined using in situ hybridisation and qPCR. Aip heterozygote and homozygote fish were assessed for growth rate and cardiac function using transmission microscopy and morphological analysis in Image J. Antisense in situ hybridisation with probes to growth hormone 1 (gh1), prolactin (prl) and proopiomelanocortin a (pomca) was used to assess pituitary phenotypes.

Results: Zebrafish aip showed 78.8% of homology to human AIP with conservation of all known interacting sites. Aip mRNA was expressed throughout the developing zebrafish embryo until 28 hours post fertilisation (hpf) stage when it became more strongly expressed in the head. Aip loss-of-function mutant animals showed reduced AIP expression. No significant differences in gross morphology were observed at 48 hpf. We report preliminary morphological characterisation of growth rate and cardiac function from 24 hpf to 7 days post fertilisation and development of the pituitary from 48 hours.

Conclusion: We have generated an aip loss-of-function zebrafish line providing ideal opportunity to study pituitary phenotype and cardiac phenotype.