Date of Award


Degree Name

Doctor of Philosophy


Molecular, Cellular, and Systemic Physiology

First Advisor

Ellsworth, Buffy


Various mouse models have allowed for identification of several transcription factors that are necessary for pituitary development. Lesions in the transcription factor genes result in pituitary hormone deficiency. Hormone deficiencies occur in approximately one in 4000 live births. Pituitary hormone deficiency may occur due to loss of a single hormone causing isolated hormone deficiency or several hormones leading to combined pituitary hormone deficiency (CPHD). Defects in genes such as LHX3, LHX4, RPX, PROP1, and PIT1 are known to contribute to CPHD in humans. FOXO1 is a member of a large family of forkhead transcription factors. FOXO1 is expressed in various tissues where it functions to regulate metabolism, maintenance of cell differentiation, vascular development, cell cycle progression and apoptosis. Previous studies in our laboratory found that FOXO1 is expressed in different subsets of pituitary cells during embryonic pituitary development, with almost 50% of GH positive somatotropes also immunopositive for FOXO1. However, the roles of FOXO1 during pituitary development have not been extensively explored. Therefore, this research focuses on the contributions of FOXO1 to pituitary development and exploration of FOXO1 as a candidate gene for CPHD. In this study, a mouse model (Foxo1 cKO) is used wherein the Foxo1 gene has been deleted in the pituitary gland by Cre-LoxP recombination system. First, expression of several genes was examined that might be associated with loss of FOXO1 in the pituitary with an aim to place FOXO1 within the hierarchy of transcription factors critical for pituitary development. The early pituitary organizers, PITX2, PITX3, LHX3, LHX4, are not affected due to deletion of Foxo1 suggesting that FOXO1 is not critical for the initial induction of oral ectoderm to form Rathke’s pouch during the early stages of pituitary development. PIT1 marks the progenitors committed to becoming somatotropes, thyrotropes or lactotropes. No apparent difference in Pit1 mRNA level as well as PIT1 immunostaining between cKO and wildtype embryos suggests that FOXO1 does not affect the commitment of progenitors to cells of the PIT1 lineage. The most significant effects of Foxo1 deletion in the pituitary gland was observed in somatotrope differentiation. There was a drastically decreased mRNA level of Ghrhr, a marker of terminally differentiated somatotropes as well as reduced expression of Neurod4 in Foxo1 cKO embryos compared to wildtype littermates. NEUROD4 is downstream of FOXO1. Another study suggests that Neurod4 deletion in the pituitary gland affects maturation of somatotrope while preserving other cell types of anterior pituitary. NEUROD4 is essential for expression of Ghrhr during embryonic development as Neurod4 deletion results in fewer somatotropes and a complete lack of Ghrhr. Therefore, it can be implied that NEUROD4 may act as an intermediate in FOXO1 mediated terminal somatotrope differentiation and loss of FOXO1 in pituitary tissue is impeding somatotrope differentiation. We also assessed the functional consequences of loss of FOXO1 in postnatal mice. The delay in differentiation of somatotropes that was evident during embryonic development seems to have recovered by P10. Therefore, we suggest that FOXO1 is important for somatotrope differentiation embryonically. FOXO1 is important for somatotrope function postnatally also. Gh1 expression, GH pituitary content and serum IGF1 levels are significantly reduced at P21. However, the cKO mice do not exhibit any growth deficit indicating that FOXO1 is dispensable for postnatal somatotrope expansion and growth. Our results show that the embryonic somatotrope phenotype associated with deletion of Foxo1 does not result in any morphological changes in postnatal cKO mice. A gene expression profiling study was done to ascertain the changes in transcriptome of the embryonic pituitary lacking FOXO1. We identified Slc25a33 and Deptor as differentially expressed genes. SLC25A33 is involved with transport of pyrimidine nucleotides across mitochondrial membrane and such transport is essential for mitochondrial DNA and RNA metabolism. Studies involving overexpression of Slc25a33 in human cells have shown it enhances cell size and mitochondrial thymidine triphosphate level but decreases ROS. Its knockdown causes depletion of mitochondrial DNA, reduced oxidative phosphorylation, cell size, and mitochondrial UTP levels, and increased ROS levels. SLC25A33 essentially maintains mitochondrial function as it regulates mitochondrial DNA replication and the ratio of transcription of mitochondrial genes relative to nuclear genes. DEPTOR acts as an intermediate in BAF60c-induced AKT activation that results in a metabolic switch from oxidative phosphorylation to glycolysis in fast-twitch myofiber. Such a switch is considered to protect mice from diet induced insulin resistance and glucose intolerance in diabetic state. In differentiating cells, significant oxidative damage occurs, which can be attributed to higher mitochondrial activity. Embryonic stem cells undergoing differentiation exhibit increased mitochondrial activity associated with mitochondrial DNA replication to encode mitochondrial electron transport chain components. During osteogenic differentiation, there is a significant increase in expression of Slc25a33, with concomitant increase in mitochondrial oxygen consumption. Similar changes have been reported during spontaneous embryonic stem cell differentiation with increase in ROS production associated with differentiation. Identification of Slc25a33 and Deptor brings to our attention, a possible mechanism which might explain the delayed somatotrope differentiation phenotype in Foxo1 cKO pituitary. We hypothesize that loss of FOXO1 and resulting suppression of Slc25a33 and Deptor results in perturbation in mitochondrial replication and imbalances ROS homeostasis, which thereby affects mitochondrial function. This hinders the somatotrope’s ability to switch to more energetically demanding metabolic pathways that are essential during terminal differentiation. A metabolic switch may be the key to the terminal differentiation of mature somatotropes from their committed progenitor cells. Our findings thus provide new insights and opens avenues for future research to investigate mechanisms of somatotrope development.




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