Using CRISPRi to investigate the role of pitx1 during pituitary gland development

The pituitary gland is an essential part of the body, as it bridges the central nervous system and the endocrine system. It governs various, essential functions and processes, such as energy metabolism, circadian rhythm, somatic growth etc. One gene, pitx1, is indicated to be expressed during early pituitary development. In mice, a knockout (KO) of Pitx1 leads to crippled hindlimbs and is lethal to the developing embryo. In zebrafish, a pitx1 KO shows no obvious phenotype, yet the larvae die at approximately 10 dpf. In this study, we wanted to investigate if pitx1 plays a potential role in the pituitary gland development of zebrafish. Contrary to a previous report, we detected no transcript of pitx1 in the early stages of pituitary gland development, yet we were able to detect a pitx1 expression in the lateral plate mesoderm of the developing embryo, in an area that will later give rise to the hindlimbs. We used both dCas9-KRAB and Cas13d to knockdown (KD) the transcription of pitx1. While we saw no significant difference in transcription, we were able to observe that a pitx1 KD increases the number of somatotropes present in the adenohypophysis and the amount of gh1 transcript, detectable by whole mount in situ hybridization. Following this, we were able to observe a significant effect of the increased gh1 expression on the growth rate of zebrafish larvae. This study provides preliminary evidence of a potential involvement of pitx1 in pituitary development and somatotrope differentiation, as well as discussing ways to optimize the CRISPRi system, which was used.