Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing and mature dopaminergic neurons. Recent studies have demonstrated that Nurr1 is essential for the induction of phenotypic markers of ventral mid-brain dopaminergic neurons whose generation is specified by the f loor plate-derived morphogenic signal sonic hedgehog (SHH), but the precise role of Nurr1 in this differentiative pathway has not been established. To provide further insights into the role of Nurr1 in the final differentiation pathway, we have examined the fate of dopamine cell precursors in Nurr1 null mutant mice. Here we demonstrate that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons. In the absence of Nurr1, neuroepithelial cells that give rise to dopaminergic neurons adopt a normal ventral localization and neuronal phenotype characterized by expression of the homeodomain transcription factor and mesencephalic marker, Ptx-3, at embryonic day 11.5. However, these late precursors fail to induce a dopaminergic phenotype, indicating that Nurr1 is essential for specifying commitment of mesencephalic precursors to the full dopaminergic phenotype. Further, as development progresses, these mid-brain dopamine precursor cells degenerate in the absence of Nurr1, resulting in loss of Ptx-3 expression and a concomitant increase in apoptosis of ventral midbrain neurons in newborn null mutant mice. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late dopaminergic precursor neurons into a complete dopaminergic phenotype.

We identified the LIM homeodomain transcription factor Lmx1b in the mesencephalic dopamine (mesDA) systems of embryos and adults. Analysis of spatiotemporal expression in Lmx1b null mutants and wild-type mice implicated a cascade involving Lmx1b in the early development of mesDA neurons. Although disruption of this cascade did not block induction of tyrosine hydroxylase (TH), a key enzyme in DA synthesis, or Nurr1, a nuclear hormone receptor, Lmx1b knockout mice failed to induce the mesDA-specific homeodomain gene Ptx3 in TH-positive neurons. Eventually, this small set of TH-positive neurons was lost during embryonic maturation. The data suggest that at least two molecular cascades operate during the specification of the mesDA system, one specifying neurotransmitter phenotype and another essential for other aspects of mesDA neuron differentiation.

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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