Loss- and gain-of-function analyses reveal targets of Pax6 in the developing mouse telencephalon.

Appropriate neurogenesis and patterning of the forebrain requires the transcription factor Pax6, yet it is largely unknown how Pax6 exerts its effects at the molecular level. To characterize Pax6-mediated regulation of gene expression during murine forebrain neurogenesis, we performed microarray analysis with tissue from the dorsal Pax6-dependent telencephalon and the ventral Pax6-negative telencephalon at the onset of neurogenesis (E12) and at mid-neurogenesis (E15) in wild-type and Pax6-deficient mutant littermates. In the Pax6-deficient cortex the expression levels of various transcription factors involved in neurogenesis (like Satb2, Nfia, AP-2gamma, NeuroD6, Ngn2, Tbr2, Bhlhb5) and the retinoic acid signalling molecule Rlbp1 were reduced. Regulation by Pax6 could be confirmed upon electroporation of a Pax6- and a dominant-negative Pax6-containing vector into embryonic cortex. Taken together, our data reveal novel insights into the molecular pathways regulated by Pax6 during cortical neurogenesis. Most intriguingly, this analysis revealed time- and region-specific differences in Pax6-mediated transcription, explaining the specific function of Pax6 at early and later stages of neurogenesis.

BMPs, FGF8 and Wnts regulate the differentiation of locus coeruleus noradrenergic neuronal precursors.

In the present study, we investigated the involvement of rhombomere 1 patterning proteins in the regulation of the major noradrenergic centre of the brain, the locus coeruleus. Primary cultures of rat embryonic day 13.5 locus coeruleus were treated with fibroblast growth factor-8, noggin and members of the bone morphogenetic and Wnt protein families. We show that bone morphogenetic proteins 2, 5 and 7 increase and noggin decreases the number of tyrosine hydroxylase-positive locus coeruleus neurons. Interestingly, from all Wnts expressed in the first rhombomere by embryonic day 12.5 in the mice, we only found expression of wnt5a mRNA in the vicinity of the locus coeruleus. In agreement with this finding, from all Wnts studied in vitro, only Wnt5a increased the number of tyrosine hydroxylase-positive neurons in locus coeruleus cultures. Finally, we also found that fibroblast growth factor-8 increased the number of tyrosine hydroxylase-positive cells in locus coeruleus cultures. Neither of the identified factors affected the survival of tyrosine hydroxylase-positive locus coeruleus noradrenergic neurons or the proliferation of their progenitors or neurogenesis. Instead, our results suggest that these patterning signals of rhombomere 1 may work to promote the differentiation of noradrenergic progenitors at later stages of development.

BMP-2 and cAMP elevation confer locus coeruleus neurons responsiveness to multiple neurotrophic factors.

The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin-3 (NT-3) and also glial cell line- derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum-free conditions, none of the factors tested, including NT-3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein-2 (BMP-2), promoted the survival of tyrosine hydroxylase (TH)-immunoreactive neurons at 6 days in vitro. However, when BMP-2 was coadministered with any of these factors the number of LC TH-positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT-3, bFGF, or BMP-2. The strongest effect (a fourfold increase in the number of TH-positive cells) was induced by cotreatment with forskolin, BMP-2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors.

Neurturin is a neuritogenic but not a survival factor for developing and adult central noradrenergic neurons.

Noradrenergic neurons of the locus coeruleus (LC) express the receptor tyrosine kinase c-ret, which binds ligands of the glial cell line-derived neurotrophic factor (GDNF) family. In the present study, we evaluated the function of neurturin (NTN), a GDNF family ligand whose function on LC neurons is unknown. Interestingly, we found that tyrosine hydroxylase (TH)-positive neurons in the LC express both GFRalpha1 and 2 receptors in a developmentally regulated fashion, suggesting a function for their preferred ligands: GDNF and NTN, respectively. Moreover, our results show that NTN mRNA expression is developmentally down-regulated in the LC and peaks in the postnatal hippocampus and cerebral cortex, during the target innervation period. In order to examine the function of NTN, we next performed LC primary cultures, and found that neither GDNF nor NTN promoted the survival of TH-positive neurons. However, both factors efficiently induced neurite outgrowth in noradrenergic neurons (147% and 149% over controls, respectively). Similarly, grafting of fibroblast cell lines engineered to express high levels of NTN did not prevent the loss of LC noradrenergic neurons in a 6-hydroxydopamine (6-OHDA) lesion model, but induced the sprouting of TH-positive cells. Thus our findings show that NTN does not promote the survival of LC noradrenergic neurons, but induces neurite outgrowth in developing noradrenergic neurons in vitro and in a model of neurodegeneration in vivo. These data, combined with data in the literature, suggest that GDNF family ligands are able to independently regulate neuronal survival and/or neuritogenesis.

Crucial role of TrkB ligands in the survival and phenotypic differentiation of developing locus coeruleus noradrenergic neurons.

The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1) the lack of alterations in double Gdnf/Nt3 null mutant mice; and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this, treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells, our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.