MON-712 Restoration of Growth and Fertility in Zebrafish (Danio Rerio) Model with PROP1 Knockout Generated by CRISPR/Cas9 Genomic Editing
Authors: Bissegatto Débora Delmonte , Fernandes Bianca Helena Ventura , da Silva Caroline Caetano , Chen Wenbiao , Junqueira Mara de Souza , Carvalho Luciani Renata Silveira 
Published: May 2020

Abstract
Introduction: Hypopituitarism is defined as the deficiency of one or more pituitary hormones and can occur due to pathogenic allelic variants in transcription factors involved in pituitary development. PROP1 gene is responsible for progenitor cell migration from the marginal zone to the anterior lobe, and its terminal differentiation into corticotropes and gonadotropes cell lines besides somatotropes, lactotropes and thyrotropes due to POU1F1 (also known as PIT1) activation. In humans, mutations in the PROP1 gene are the most common cause of congenital hypopituitarism with GH, TSH, LH/FSH, and progressive ACTH deficiencies. A dwarf phenotype with short stature, pituitary hormone deficiency, and infertility has been described in humans and Ames mice lineage harboring mutations in the PROP1/Prop1 gene. Another valuable animal model used in basic research is the zebrafish (Danio rerio) due to a high homology in neuroendocrine functioning. To test the potential of this model, in our previous study, a 32bp insertion carrying a stop codon was directed into the second exon of prop1 with CRISPR/Cas9, establishing a homozygous mutant strain (prop1mut). Objective: To characterize the phenotype and expression patterns of transcription factors and hormones in the zebrafish prop1mut lineage. Methods: prop1, pit1, and gh1 mRNA levels were analyzed during embryonic development at 24 and 72 hours post-fertilization (hpf). RNA from 30 pooled embryos was extracted using DirectZol RNA Miniprep. cDNA was synthesized from 1ug of total RNA using High-Capacity cDNA Reverse Transcription Kit and qPCR was performed using SYBR Green PCR Master Mix. Gene expression was normalized to ef1a and the prop1mut group was compared with the control wild type group (WT). Animals were kept in the tanks at a density of 15 animals/liter and images were acquired at 13 and 20 days post fertilization (dpf) after brief anesthetization using a stereomicroscope and measured in ImageJ software to determine the larval standard length from nose to the end of the spinal cord. Results: At 24 and 72hpf, prop1mut embryos expressed the altered prop1 mRNA at similar levels to the prop1 expression observed in WT. Lower pit1 expression in prop1mut embryos was observed at both periods (p&lt;0.01). Albeit in low levels, similar gh1 expression was observed in both lineages at 24hpf, and prop1mut embryos presented lower gh1 expression at 72hpf (p&lt;0.001). prop1mut larvae presented a significant decrease in size at 13dpf (p&lt;0.001) but not at 20dpf. Conclusion: In this study, the prop1mut zebrafish model exhibited a dwarf phenotype during larval development associated with diminished pit1 and gh1 expression during the embryonic stage. Additionally, in the juvenile stage, the development rate in prop1mut animals was restored, presenting similar standard lengths observed in WT animals.