The data provide essential new information toward understanding the complex interactions required for normal development and function of the mammalian eye. The results also underscore the importance of confirming neural crest and mesoderm fates in a model mammalian system. The complementary systems used in this study should be useful for studying the respective cell fates in other organ systems.

Three major PITX2 isoforms are differentially expressed in human, mice, zebrafish, chick, and frog tissues. To demonstrate differential regulation of gene expression by these isoforms we used three different promoters and three cell lines. Transient transfection of Chinese hamster ovary, HeLa, and LS-8 cell lines revealed differences in PITX2A and PITX2C activation of the PLOD1 and Dlx2 promoters, however, PITX2B is inactive. In contrast, PITX2B actives the pituitary-specific Prolactin promoter at higher levels than either PITX2A or PITX2C. Interestingly, co-transfection of either PITX2A or PITX2C with PITX2B results in a synergistic activation of the PLOD1 and Dlx2 promoters. Furthermore, PITX2 isoforms have different transcriptional activity dependent upon the cells used for transfection analysis. We have isolated a fourth PITX2 isoform (PITX2D) expressed only in humans, which acts to suppress the transcriptional activity of the other PITX2 isoforms. Electrophoretic mobility shift assays and glutathione S-transferase pull-down experiments demonstrated that all isoforms interact with PITX2D and that PITX2B forms heterodimeric complexes with PITX2A and PITX2C. Our research provides a molecular basis for differential gene regulation through the expression of PITX2 isoforms. PITX2 isoform activities are both promoter-and cell-specific, and our data reveal new mechanisms for PITX2-regulated gene expression.

The specification and differentiation of dentate gyrus granule neurons in the hippocampus require temporally and spatially coordinated actions of both intrinsic and extrinsic molecules. The basic helix-loop-helix transcription factor Neurogenin2 (Ngn2) and NeuroD1 are key regulators in these processes. Based on existing classification, we analyzed the molecular events occurring during hippocampal neurogenesis, primarily focusing on juvenile animals. We found that Ngn2 is transiently expressed by late type-2a amplifying progenitors. The Ngn2 progenies mature into hippocampal granule neurons. Interestingly, the loss of Ngn2 at early stages of development leads to a robust reduction in neurogenesis, but does not disturb granule neuron maturation per se. We found that the role of Ngn2 is to maintain progenitors in an undifferentiated state, allowing them to amplify prior to their maturation into granule neurons upon NeuroD1 induction. When we overexpressed Ngn2 and NeuroD1 in vivo, we found NeuroD1 to exhibit a more pronounced neuron-inductive effect, leading to granule neuron commitment, than that displayed by Ngn2. Finally, we observed that all markers expressed during the transcriptional control of hippocampal neurogenesis in rodents are also present in the human hippocampus. Taken together, we demonstrate a critical role of for Ngn2 and NeuroD1 in controlling neuronal commitment and hippocampal granule neuroblast formation, both during embryonic development and in post-natal hippocampal granule neurogenesis.

Spinal cholinergic neurons are critical for motor function in both the autonomic and somatic nervous systems and are affected in spinal cord injury and in diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy. Using two screening approaches and in situ hybridization, we identified 159 genes expressed in typical cholinergic patterns in the spinal cord. These include two general cholinergic neuron markers, one gene exclusively expressed in motor neurons, and nine genes expressed in unknown subtypes of somatic motor neurons. Further, we present evidence that chondrolectin (Chodl) is expressed by fast motor neurons and that estrogen-related receptor beta (ERRbeta) is a candidate marker for slow motor neurons. In addition, we suggest paired-like homeodomain transcription factor 2 (Pitx2) as a marker for cholinergic partition cells.

The PITX family of transcription factors regulate the development of many organs. Pitx1 mutants have a mild pituitary phenotype, but Pitx2 is necessary for the development of Rathke's pouch, expression of essential transcription factors in gonadotropes, and expansion of the Pit1 lineage. We report that lack of Pitx2 causes the pouch to undergo excessive cell death, resulting in severe pituitary hypoplasia. Transgenic overexpression of PITX2 in the pituitary can increase the gonadotrope population, suggesting that the absolute concentration of PITX2 is important for normal pituitary cell lineage expansion. We show that PITX1 and PITX2 proteins are present in similar expression patterns throughout pituitary development and in the mature pituitary. Both transcription factors are preferentially expressed in adult gonadotropes and thyrotropes, suggesting the possibility of overlap in maintenance of adult pituitary functions within these cell types. Double knockouts of Pitx1 and Pitx2 exhibit severe pituitary hypoplasia and fail to express the transcription factor LHX3. This indicates that these PITX genes are upstream of Lhx3 and have compensatory roles during development. Thus, the combined dosage of these PITX family members is vital for pituitary development, and their persistent coexpression in the adult pituitary suggests a continued role in maintenance of pituitary function.