A homeodomain gene Ptx 3 has highly restricted brain expression in mesencephalic dopaminergic neurons

Abstract

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...

Pitx3 is required for development of substantia nigra dopaminergic neurons

“…Pitx3 gene expression is restricted to the developing eye and DA progenitor cells from embryonic day 11 throughout adult life in mice (13,14). In the brain, Pitx3 mRNA localizes specifically to the SN and VTA (14). A reduction in Pitx3 mRNA levels is observed in the ventral midbrain of Lmx1b knockout mice, 6-hydroxydopamine-lesioned rats, and Parkinson's patients (8,11,12,14).…”

“…In the brain, Pitx3 mRNA localizes specifically to the SN and VTA (14). A reduction in Pitx3 mRNA levels is observed in the ventral midbrain of Lmx1b knockout mice, 6-hydroxydopamine-lesioned rats, and Parkinson's patients (8,11,12,14). Yet, Pitx3 expression is maintained in the ventral midbrain of Nurr1 null mutant embryos (12).…”

“…Pitx3 gene expression is restricted to the developing eye and DA progenitor cells from embryonic day 11 throughout adult life in mice (13,14). In the brain, Pitx3 mRNA localizes specifically to the SN and VTA (14).…”

Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon

“…Several developmental genes, such as Engrailed (En) 1 and 2, and Lmx1b, and cellular proteins such as aldehyde dehydrogenase-1 (Aldh1, also known as AHD-2), have been shown to be expressed in DA neuron precursors in the developing VM. However, the expression of these proteins is not restricted to the precursor pool, but is maintained also in the differentiated, post-mitotic mesDA neurons (9,14,21,30,32,37). Here we report that the proneural gene product Neurogenin2 (Ngn2) within the VM is expressed exclusively in the part of the proliferating ventricular zone (VZ), which gives rise to the migrating mesDA neuron precursors, but not in the differentiated, tyrosine hydroxylase (TH)-expressing postmitotic mesDA neurons.…”

Motor deficits and altered striatal gene expression in aphakia (ak) mice

“…Pitx3 is a member of the pituitary family of bicoid type homeobox transcription factors (Semina et al, 1997). It is expressed in midbrain dopaminergic neurons by E11.5 (Smidt et al, 1997), where it facilitates differentiation of the A9 cell group constituting the substantia nigra pars compacta (SNc; Chung et al, 2005). In homozygous ak mice, genomic deletions within the promoter and exon1 of the Pitx3 gene (Rieger et al, 2001;Semina et al, 2000) result in incomplete lens development and aphakia.…”