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Analyses were performed with ClustalW2.0 and rVISTA. Additional transcription EPLG1 factor binding sites within the Core R3 included putative elements for CCAAT enhancer binding protein (CEBP) and SOX2 sites (Fig. this sequence, tandem binding sites recognized by the islet-1 (ISL1) LIM-HD protein are essential for enhancer activity in the pituitary and spine, and a pituitary homeobox 1 (PITX1) bicoid class HD element is required for spatial patterning in the developing pituitary. This study establishes ISL1 as a novel transcriptional regulator ofLHX3and describes a potential mechanism for regulation by PITX1. Moreover, these studies suggest models for analyses of the transcriptional pathways coordinating the expression of other LIM-HD genes and provide tools for the molecular analysis and genetic counseling of pediatric patients with combined pituitary hormone deficiency. Studies of animal models and human patients have established that Lin-11, Isl-1, Mec-3 (LIM)-homeodomain (HD) genes encode regulatory transcription factors that are critical for the development of specialized cells in many tissues and organs, including the nervous system, skeletal muscle, the heart, the kidneys, and endocrine organs, such as the pancreas and the pituitary gland (1). The pituitary secretes hormones that control key developmental and physiological processes, including the stress response, reproduction, metabolism, growth, and lactation. The gland has dual origins with the posterior pituitary lobe originating from the neuroectoderm or diencephalon and the anterior and intermediate lobes (IL) developing from the oral ectoderm. Signaling gradients between the diencephalon and oral ectoderm result in invagination of the oral ectoderm, forming Rathke’s pouch, the primordium of the anterior/IL (2). Subsequent cellular determination, differentiation, and proliferation events are controlled in part by multiple transcription factors to establish the specialized hormone-secreting cell types of the pituitary (reviewed in Ref.3). The five main hormone-secreting cell types found in the anterior pituitary are the corticotropes, gonadotropes, thyrotropes, Theophylline-7-acetic acid somatotropes, and lactotropes secreting ACTH, FSH and LH, thyroid-stimulating hormone (thyrotropin), GH, and prolactin, respectively. The LHX3 LIM-HD transcription factor is required for the formation of the anterior pituitary gland, including specification of somatotropes, thyrotropes, lactotropes, and gonadotropes, and has additional roles in the development of spinal motoneurons (reviewed in Ref.4). The defects displayed byLhx3knockout mice and patients with coding region mutations in theLHX3gene emphasize the importance of LHX3 proteins in pituitary development.Lhx3knockout mice die shortly after birth. In these animals, Rathke’s pouch forms but fails to effectively develop, lacking four of the five differentiated cell types, with only a residual population of corticotropes Theophylline-7-acetic acid being observed (5). To date, eleven homozygous mutations in the protein-coding regions of humanLHX3have been identified that result in combined pituitary hormone deficiency (CPHD) syndrome. Although some patients withLHX3mutations survive, they are short in stature and have hypothyroidism and hypogonadism. These deficiencies are similar to the abnormal pituitary development and hormone losses seen in theLhx3knockout mice (5). Development of the nervous system also is affected, causing loss of normal neck rotation, developmental delay, and deafness in some cases Theophylline-7-acetic acid (611). The mammalianLHX3genes have seven coding exons and six introns and produce two major mRNA, calledLHX3aandLHX3b. These RNA are transcribed from two TATA-less, GC-rich promoters located upstream of exons Ia and Ib that are recognized by specificity protein-1 and nuclear factor I (12). Several upstream factors, including fibroblast growth factor 8, pituitary homeobox (PITX)1, PITX2, sex-determining region Theophylline-7-acetic acid Y-box (SOX)2, LHX4, and forkhead box P1 have been implicated in the regulation ofLHX3transcription in pituitary and neural tissues. The expression of fibroblast growth factor 8 in the adjacent diencephalon and Rathke’s pouch is required for the activation of mouseLhx3andLhx4(2).Pitx1/Pitx2double knockout mice fail to expressLhx3and have a similar phenotype toLhx3-null mice (13). LHX3 protein expression is maintained inPitx1-null andPitx2-null mice, suggesting an overlapping function of PITX1 and PITX2 with expression of either sufficient to activateLhx3during pituitary development (14,15). SOX2 has also been shown to bind and activate.