Mechanisms underlying pituitary hypoplasia and failed cell specification in Lhx3-deficient mice

AbstractThe LIM homeodomain transcription factor, LHX3, is essential for pituitary development in mouse and man. Lhx3 engineered null mice have profound pituitary hypoplasia that we find is attributable to an increase in cell death early in pituitary development. Dying cells are localized to regions of TPIT expression indicating that cell death may contribute to the severe reduction in differentiated corticotrope cells and lower expression of the corticotrope transcription factors, TPIT and NEUROD1. Lhx3 deficiency also results in dorsal ectopic expression of transcription factors characteristic of gonadotropes, SF1 and ISL1, but no gonadotropin expression. This apparent disturbance of cell differentiation may be due, in part, to loss of NOTCH2. NOTCH2 is normally expressed in the pituitary at the boundary between dorsal, proliferating cells and ventral, differentiating cells and is important for maintaining dorsal–ventral patterning in other organs. Thus, Lhx3 contributes significantly to pituitary development by maintaining normal dorsal–ventral patterning, cell survival, and normal expression of corticotrope-specific transcription factors, which are necessary for repressing ectopic gonadotrope differentiation

The forkhead transcription factor, FOXP3: a critical role in male fertility in mice.

Fertility is dependent on the hypothalamic-pituitary-gonadal axis. Each component of this axis is essential for normal reproductive function. Mice with a mutation in the forkhead transcription factor gene, Foxp3, exhibit autoimmunity and infertility. We have previously shown that Foxp3 mutant mice have significantly reduced expression of pituitary gonadotropins. To address the role of Foxp3 in gonadal function, we examined the gonadal phenotype of these mice. Foxp3 mutant mice have significantly reduced seminal vesicle and testis weights compared with Foxp3(+/Y) littermates. Spermatogenesis in Foxp3 mutant males is arrested prior to spermatid elongation. Activation of luteinizing hormone signaling in Foxp3 mutant mice by treatment with human chorionic gonadotropin significantly increases seminal vesicle and testis weights as well as testicular testosterone content and seminiferous tubule diameter. Interestingly, human chorionic gonadotropin treatments rescue spermatogenesis in Foxp3 mutant males, suggesting that their gonadal phenotype is due primarily to a loss of pituitary gonadotropin stimulation rather than an intrinsic gonadal defect

Transformation of Soybean with an Antisense Cholinephosphotransferase Gene

Cholinephosphotransferase is the enzyme which forms phosphatidylcholine, a major membrane lipid. The goal of this project was to produce a transgenic soybean which contained a modified cholinephosphotransferase gene in the antisense orientation. Antisense genes express the reverse complement of an mRNA. When \u27this copy hybridizes with the normal mRNA it blocks translation, thereby eliminating the enzyme. In the case of cholinephosphotransferase this will eliminate the substrate for polyunsaturatec;I fatty acid synthesis, resulting in seeds with high levels of saturated and monounsaturated fatty acids. A particle inflow gun was used to target the anti-sense construction into soybean embryonic tissue. - The resulting tissue was regenerated. The antisense plasmid has been constructed, and has been successfully transformed into soybeans. However, the two soybeans that tested positive for the antisense construct could not be regenerated

Functional Role of Gonadotrope Plasticity and Network Organization

Gonadotrope cells of the anterior pituitary are characterized by their ability to mount a cyclical pattern of gonadotropin secretion to regulate gonadal function and fertility. Recent in vitro and in vivo evidence suggests that gonadotropes exhibit dramatic remodeling of the actin cytoskeleton following gonadotropin-releasing hormone (GnRH) exposure. GnRH engagement of actin is critical for gonadotrope function on multiple levels. First, GnRH-induced cell movements lead to spatial repositioning of the in vivo gonadotrope network toward vascular endothelium, presumably to access the bloodstream for effective hormone release. Interestingly, these plasticity changes can be modified depending on the physiological status of the organism. Additionally, GnRH-induced actin assembly appears to be fundamental to gonadotrope signaling at the level of extracellular signal-regulated kinase (ERK) activation, which is a well-known regulator of luteinizing hormone (LH) β-subunit synthesis. Last, GnRH-induced cell membrane projections are capable of concentrating LHβ-containing vesicles and disruption of the actin cytoskeleton reduces LH secretion. Taken together, gonadotrope network positioning and LH synthesis and secretion are linked to GnRH engagement of the actin cytoskeleton. In this review, we will cover the dynamics and organization of the in vivo gonadotrope cell network and the mechanisms of GnRH-induced actin-remodeling events important in ERK activation and subsequently hormone secretion

Forkhead Box O1 is present in quiescent pituitary cells during development and is increased in the absence of p27 Kip1.

Congenital pituitary hormone deficiencies have been reported in approximately one in 4,000 live births, however studies reporting mutations in some widely studied transcription factors account for only a fraction of congenital hormone deficiencies in humans. Anterior pituitary hormones are required for development and function of several glands including gonads, adrenals, and thyroid. In order to identify additional factors that contribute to human congenital hormone deficiencies, we are investigating the forkhead transcription factor, FOXO1, which has been implicated in development of several organs including ovary, testis, and brain. We find that FOXO1 is present in the nuclei of non-dividing pituitary cells during embryonic development, consistent with a role in limiting proliferation and/or promoting differentiation. FOXO1 is present in a subset of differentiated cells at e18.5 and in adult with highest level of expression in somatotrope cells. We detected FOXO1 in p27(Kip1)-positive cells at e14.5. In the absence of p27(Kip1) the number of pituitary cells containing FOXO1 is significantly increased at e14.5 suggesting that a feedback loop regulates the interplay between FOXO1 and p27(Kip1)

Loss of Foxm1 Results in Reduced Somatotrope Cell Number during Mouse Embryogenesis.

FOXM1, a member of the forkhead box transcription factor family, plays a key role in cell cycling progression by regulating the expression of critical G1/S and G2/M phase transition genes. In vivo studies reveal that Foxm1 null mice have a 91% lethality rate at e18.5 due to significant cardiovascular and hepatic hypoplasia. Thus, FOXM1 has emerged as a key protein regulating mitotic division and cell proliferation necessary for embryogenesis. In the current study, we assess the requirement for Foxm1 in the developing pituitary gland. We find that Foxm1 is expressed in the pituitary at embryonic days 10.5-e18.5 and localizes with markers for active cell proliferation (BrdU). Interestingly, direct analysis of Foxm1 null mice at various embryonic ages, reveals no difference in gross pituitary morphology or cell proliferation. We do observe a downward trend in overall pituitary cell number and a small reduction in pituitary size in e18.5 embryos suggesting there may be subtle changes in pituitary proliferation not detected with our proliferation makers. Consistent with this, Foxm1 null mice have reductions in both the somatotrope and gonadotrope cell populations

FOXO1 is present in the nuclei of pituitary cells at an increasing frequency as development progresses.

<p>Immunohistochemistry for FOXO1 (green) was performed on midsagittal pituitary sections. (A) FOXO1 is present in the developing pituitary by e10.5. Nuclear FOXO1 is apparent where the invaginating Rathke’s pouch is joined to the oral ectoderm that will form the mouth (see inset). (B) By e12.5 FOXO1 is present almost entirely in the cytoplasm of pituitary cells. (C) A few pituitary cells contain nuclear FOXO1 at e14.5. (D) At e18.5 the developing pituitary contains a mostly nuclear FOXO1. (E) In adults, FOXO1 is present in the anterior and intermediate lobes of the pituitary gland, but not in the posterior lobe (data not shown). In the adult pituitary FOXO1 is primarily nuclear (inset, arrow). Some cytoplasmic FOXO1 (inset, arrow head) is also present. (F) Immunohistochemistry for FOXO1. (G) Beta-galactosidase staining of pituitary from <i>Foxo1^+/LacZ</i> mice identifies cells in which the endogenous <i>Foxo1</i> promoter is active. (H) An overlay of immunohistochemical staining for FOXO1 (green) and β-galactosidase staining of pituitary from <i>Foxo1^+/LacZ</i> mice (blue). (F–H) Arrows highlight examples of co-localized cells. Pictures are taken at 200X (A–E) or 630X (F–H). Insets are magnified 600X. Scale bars represent 100 µm. All cell nuclei were marked with DAPI (A–E, blue). Oral ectoderm (OE), infundibulum (INF), ventral diencephalon (VD), Rathke’s pouch (RP), posterior lobe (PL), intermediate lobe (IL), anterior lobe (AL).</p