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The pituitary gland contains six distinct hormone-producing cell types that arise sequentially during organogenesis. The first cells to differentiate are those that express the pro-opiomelanocortin (POMC) gene in the anterior pituitary lobe. The other lineages, which appear later, include cells that are dependent on the POU factor Pit-1 and another POMC-expressing lineage in the intermediate pituitary lobe. Using AtT-20 cells as a model for early expression of POMC in the anterior pituitary, we have defined a regulatory element conferring cell specificity of transcription and cloned a cognate transcription factor. This factor, Ptx1 (pituitary homeo box 1), contains a homeo box related to those of the anterior-specific genes bicoid and orthodenticle in Drosophila, and Otx-1 and Otx-2 in mammals. Ptx1 activates transcription upon binding a sequence related to the Drosophila bicoid target sites. Ptx1 is the only nuclear factor of this DNA-binding specificity that is detected in AtT-20 cells, and it is expressed at high levels in a subset of adult anterior pituitary cells that express POMC. However, Ptx1 is expressed in most cells of Rathke's pouch at an early time during pituitary development and before final differentiation of hormone-producing cells. Thus, Ptx1 may have a role in differentiation of pituitary cells, and its early expression pattern suggests that it may have a role in pituitary formation. In the adult pituitary gland, Ptx1 appears to be recruited for cell-specific transcription of the POMC gene.

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxydopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.The patterning of the developing mammalian brain is thought to involve cascades of signaling molecules and transcription factors, but the mechanisms for generation of distinct neuronal cell types during terminal differentiation are still largely speculative (1, 2). Yet, the specification of individual neuronal phenotypes underlies the assembly of neural circuits essential for brain function. The mesencephalic dopaminergic (mesDA) system consists of a limited set of neurons that are well defined anatomically and functionally (3-5). Their specific degeneration in Parkinson disease reveals their functional properties in control of behavior and movement as well as a unique vulnerability (6-10). In a search for homeobox genes associated with a unique neuronal lineage, we isolated a cDNA encoding a bicoid-related homeobox gene Ptx3, a member of the Ptx subfamily (11)(12)(13)(14). This gene is strictly expressed in mesDA neurons. METHODS AND MATERIALSCloning of Ptx3 Gene Transcripts. Poly(A) ϩ RNA from hypothalamic fragments of the adult rat brain were subjected to reverse transcriptase-PCR with primers based on brainexpressed homeobox genes: upstream, 5Ј-GMRSCGM-SAVMGSACMMBCTTYAC-3Ј; downstream, 5Ј-TGGT-TYMRVAAYCGYHGMGCMARRTG-3Ј. The annealing temperature was 40°C. The PCR product was used to screen an adult rat hypothalamus library in gt11. Isolated phage DNA was cut with EcoRI, the insert of Ϸ1.2 kb was subcloned into pGEM7Zf(ϩ), and both strands of the insert were sequenced.Northern Analysis. Total RNA extracted from tissues of the adult rat by Rnazol (Biotecx Laboratories, Houston) was fractionated on formaldehyde-agarose gels and transferred onto a nylon membrane (Hybond-N, Amersham) by downward capillary blotting. Blots were hybridized with a 32 P-randomprimed-labeled complete Ptx3 cDNA at 65°C overnight (15). Autoradiography was performed with a Fujix BAS1000 phosphor-imager (Fuji).Cell Culture, Transfection, and Gel Retardation Assays. Murine fibroblast L cells were grown in DMEM supplemented with 10% FCS. L cells were transfected by the calcium phosphate method (16). Precipitate containing 3 g of reporter plasmid, 1 g of effector plasmid, 1 g of RSV-human growth hormone (hGH) internal control plasmid, and 5 g of carrier DNA (pSP64, Promega) was app...

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