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Wingless-related MMTV integration site 1 (WNT1)/β-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/β-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/β-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset.Dkk3transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD.SIGNIFICANCE STATEMENTWe show here that Dickkopf 3 (DKK3), a secreted modulator of WNT (Wingless-related MMTV integration site)/β-catenin signaling, is both necessary and sufficient for the proper differentiation and survival of a rostrolateral (parabrachial pigmented nucleus and dorsomedial substantia nigra pars compacta) mesodiencephalic dopaminergic neuron subset, usingDkk3mutant mice and murine primary ventral midbrain and pluripotent stem cells. The progressive loss of these dopamine-producing mesodiencephalic neurons is a hallmark of human Parkinson's disease, which can up to now not be halted by clinical treatments of this disease. Thus, the soluble DKK3 protein might be a promising new agent for the improvement of current protocols for the directed differentiation of pluripotent and multipotent stem cells into mesodiencephalic dopaminergic neurons and for the promotion of their survivalin situ.

Neurons synthesizing the neurotransmitter dopamine exert crucial functions in the mammalian brain. The biggest and most important population of dopamine-synthesizing neurons is located in the mammalian ventral midbrain, and controls and modulates the execution of motor, cognitive, affective, motivational, and rewarding behaviours. Degeneration of these neurons leads to motor deficits that are characteristic of Parkinson's Disease, while their dysfunction is involved in the pathogenesis of psychiatric disorders including schizophrenia and addiction. Because the aetiology and therapeutic prospects for these diseases include neurodevelopmental aspects, substantial scientific interest has been focused on deciphering the mechanistic pathways that control the generation and survival of these neurons during embryonic development. Researches during the last decade revealed the pivotal role of the secreted Wnt1 ligand and its signalling cascade in the generation of the dopamine-synthesizing neurons in the mammalian ventral midbrain. Here, we summarize the initial and more recent findings that have unravelled several Wnt1-controlled genetic networks required for the proliferation and commitment of ventral midbrain progenitors to the dopaminergic cell fate during pre-midgestational embryonic stages, and for the correct differentiation of these progenitors into postmitotic dopamine-synthesizing neurons at late midgestational embryonic and fetal stages.

The isthmic organizer mediating differentiation of mid- and hindbrain during vertebrate development is characterized by a well-defined pattern of locally restricted gene expression domains around the mid-hindbrain boundary (MHB). This pattern is established and maintained by a regulatory network between several transcription and secreted factors that is not yet understood in full detail. In this contribution we show that a Boolean analysis of the characteristic spatial gene expression patterns at the murine MHB reveals key regulatory interactions in this network. Our analysis employs techniques from computational logic for the minimization of Boolean functions. This approach allows us to predict also the interplay of the various regulatory interactions. In particular, we predict a maintaining, rather than inducing, effect of Fgf8 on Wnt1 expression, an issue that remained unclear from published data. Using mouse anterior neural plate/tube explant cultures, we provide experimental evidence that Fgf8 in fact only maintains but does not induce ectopic Wnt1 expression in these explants. In combination with previously validated interactions, this finding allows for the construction of a regulatory network between key transcription and secreted factors at the MHB. Analyses of Boolean, differential equation and reaction-diffusion models of this network confirm that it is indeed able to explain the stable maintenance of the MHB as well as time-courses of expression patterns both under wild-type and various knock-out conditions. In conclusion, we demonstrate that similar to temporal also spatial expression patterns can be used to gain information about the structure of regulatory networks. We show, in particular, that the spatial gene expression patterns around the MHB help us to understand the maintenance of this boundary on a systems level., Author Summary Understanding brain formation during development is a tantalizing challenge. It is also essential for the fight against neurodegenerative diseases. In vertebrates, the central nervous system arises from a structure called the neural plate. This tissue is divided into four regions, which continue to develop into forebrain, midbrain, hindbrain and spinal cord. Interactions between locally expressed genes and signaling molecules are responsible for this patterning. Two key signaling molecules in this process are Fgf8 and Wnt1 proteins. They are secreted from a signaling center located at the boundary between prospective mid- and hindbrain (mid-hindbrain boundary, MHB) and mediate development of these two brain regions. Here, we logically analyze the spatial gene expression patterns at the MHB and predict interactions involved in the differentiation of mid- and hindbrain. In particular, our analysis indicates that Wnt1 depends on Fgf8 for stable maintenance. A time-course analysis of Wnt1 expression after implantation of Fgf8-coated beads in mouse neural plate/tube explants experimentally validates our prediction about the interactions between these two key patterning molecules. Subsequently, we demonstrate that available data allows construction of a mathematical model able to explain the maintenance of the signaling center at the MHB. We begin to understand this small aspect of brain formation on a systems level.

Background The establishment of the mid-hindbrain region in vertebrates is mediated by theisthmic organizer, an embryonic secondary organizer characterized by awell-defined pattern of locally restricted gene expression domains with sharplydelimited boundaries. While the function of the isthmic organizer at themid-hindbrain boundary has been subject to extensive experimental studies, itremains unclear how this well-defined spatial gene expression pattern, which isessential for proper isthmic organizer function, is established during vertebratedevelopment. Because the secreted Wnt1 protein plays a prominent role in isthmicorganizer function, we focused in particular on the refinement of Wnt1gene expression in this context. Results We analyzed the dynamics of the corresponding murine gene regulatory network andthe related, diffusive signaling proteins using a macroscopic model for thebiological two-scale signaling process. Despite the discontinuity arisingfrom the sharp gene expression domain boundaries, we proved the existence ofunique, positive solutions for the partial differential equation system. Thisenabled the numerically and analytically analysis of the formation and stabilityof the expression pattern. Notably, the calculated expression domain ofWnt1 has no sharp boundary in contrast to experimental evidence. Wesubsequently propose a post-transcriptional regulatory mechanism for Wnt1miRNAs which yields the observed sharp expression domain boundaries. Weestablished a list of candidate miRNAs and confirmed their expression pattern byradioactive in situ hybridization. The miRNA miR-709 was identified as apotential regulator of Wnt1 mRNA, which was validated by luciferasesensor assays. Conclusion In summary, our theoretical analysis of the gene expression pattern induction atthe mid-hindbrain boundary revealed the need to extend the model by an additionalWnt1 regulation. The developed macroscopic model of a two-scaleprocess facilitate the stringent analysis of other morphogen-based patterningprocesses.