We show here that Pbx1a is expressed in mesDA neurons from E11 into adulthood. During early embryogenesis, its expression in the neural tube is abundant, and becomes later confined in the ventral midbrain to only mesDA neurons. The co-expression of Pbx1a and Prep1 in mesDA neurons suggests that Pbx1 nuclear localization is achieved in this neuronal population through molecular association with Prep1. We, furthermore, show an aberrant mesDA axonal projection in Pbx1−/− embryos, which is likely the result of the loss of DCC expression. However we were not able to demonstrate direct Pbx1 binding on the three highly conserved Pbx1 binding sites in the first intron of DCC by ChIP.
A number of studies have shown molecular interactions between Pbx proteins and several other transcription factors and transcriptional co-regulators. The most studied Pbx partners are the Hox proteins. However, Pbx members form functional heterodimeric complexes with other homeoproteins, such as Engrailed and Pdx1, and other non-homeodomain transcription factors of the basic helix-loop-helix, forkhead and Smad family, as well as with members of the nuclear receptor superfamily [2, 6]. Pbx loss of function phenotype is very often correlated to the function of the associated partner. Pbx1-deficient mice die at E15.5, displaying severe hypoplasia (lungs, liver, stomach, gut, kidneys and pancreas), ectopia (thymus and kidneys) or aplasia (spleen, adrenal gland) of multiple organs, and widespread defects of the axial and appendicular skeleton . Although mice with Pbx1 targeted mutation exhibit some degree of homeotic transformations, they do not perfectly resemble mutants for Hox genes, their most studied partners. The same can be said for other Pbx mutants. Pbx3-deficient mice survive to term, but die soon after birth from central respiratory failure . Pbx1 and Pbx3 have overlapping embryonic expression domains and could therefore exhibit redundant functions. In contrast to Pbx1- and Pbx3-deficient mice, Pbx2-deficient mice are viable and display no apparent phenotype despite its broad expression . Therefore the phenotype of the Pbx targeted mutants could be the result of compensatory functions of other Pbx members and/or partial partner-independent functions [2, 6].
The phenotypical alterations in mesDA neurons of Pbx1-deficient mice can be considered in correlation to the well-described Engrailed phenotype in these cells. The targeted deletion of both Engrailed genes leads to severe tissue deletion in the mesencephalon and loss of mesDA neurons at birth . A more detailed analysis of these mutant mice revealed that the dopaminergic neurons are generated in the mesencephalic flexure, but die by E14 without extending axonal processes . MesDA neurons in Pbx1-deficent embryos survive beyond E14 and are able to extend axons; a phenotype that seems to diverge from the complete ablation of mesDA neurons observed in Engrailed double mutant embryos. Yet a cooperative function of Engrailed and Pbx1 cannot be excluded on the base of this sole phenotypic resemblance. Engrailed mutation show a gene-dose dependent effect on the survival of mesDA neurons  and no information have been reported about the axonal projections of mesDA neurons in other single or compound Engrailed mutants. Furthermore, our analysis does not exclude a redundant effect of other Pbx genes. The presence of Pbx3 mRNA expression in these neurons indicates the possibility of a compensatory effect in absence of Pbx1, therefore restoring the threshold Pbx proteins concentration required for a correct development.
We report here that Pbx1 loss of function leads to defasciculation and misrouting of mesDA axons in the border between di- and telencephalon. Since Pbx1 is expressed in mesDA neurons as well as in the developing target tissue , the axonal outgrowth phenotype of Pbx1-deficient mice could reflect alterations in either of the two. The unaltered expression of netrin-1 in the ganglionic eminence, the intact morphology of the tissue and the loss of DCC expression suggest that the mesDA axonal phenotype is likely attributable to a cell-autonomous function of Pbx1.
Several studies suggest that multiple cues collaborate to guide dopaminergic axons into a restricted domain through the diencephalon. Initially, migration of mesDA axons rostrally is determined by repulsion from a posterior source of semaphorin. Once in the diencephalon, mesDA axons are constrained in a narrow path established by multiple signals that keep axons from diverging ventrally or dorsally. The ventral boundary requires both Robo/Slit [48, 49] and Netrin/DCC [35, 36] opposing actions, as both slits repulsion and netrins attraction actions contribute to prevent dopaminergic axons from crossing the midline. Dorsal repulsion instead is likely mediated by attractive cues only, such as netrin and Sonic hedgehog [35, 49–51]. Finally, mesDA projections into the basal forebrain and cortex require an unusual attractive activity of semaphorin .
A recent analysis of DCC loss of function in vitro and in vivo demonstrated that DCC regulates neuronal precursor cell migration, axon guidance and axonal terminal arborization . Nevertheless, even in absence of DCC expression, mesDA axons are able to reach their target tissue . Differently from the previous report, however, in Pbx1-deficient embryos, loss of DCC expression has no effect on cell migration and seems to affect only long-range axon guidance. In Pbx1-deficent mice, axonal outgrowth is not affected until the mesDA neurons reach the border region between di- and telencephalon, and only at this point does Pbx1-mediated DCC/netrin signaling seem to be required. Unfortunately, Pbx1 mutant mice die at E15.5, preventing further analysis of the phenotype induced by the loss of DCC expression in these mice. No information is available on the embryonic phenotype of DCC mutants to be compared with those of Pbx1 mutants. Furthermore, analysis at later stages of the basal forebrain structures affected by abnormal nigro-striatal axonal targeting (dorsal striatum, olfactory tubercle, etc…) is not possible in Pbx1-mutants as complete maturation of dopaminergic innervations to the forebrain takes place between E15 and P0 [34, 53].
According to the Stein and Tessier-Lavigne ‘Hierarchical organization of guidance receptors’ model , activation of DCC by netrin, and concomitantly of Robo by Slit, leads to silencing of the attractive DCC-mediated netrin response without affecting its growth-stimulatory effect. Indeed, both DCC and Robo are expressed in mesDA neurons at developmental stages consistent with the defect observed in Pbx1 mutant embryos and could contribute to the observed phenotype [35, 36, 49]. Furthermore, a recent study indicated that loss of Slit/Robo signaling leads to widespread errors in mesDA axonal trajectories in the diencephalon, similar to those observed in Pbx1-deficient mice .