Nilima Prakash

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinsons disease.

Genetic networks controlling the development of midbrain dopaminergic neurons

Recent data have substantially advanced our understanding of midbrain dopaminergic neuron development. Firstly, a Wnt1‐regulated genetic network, including Otx2 and Nkx2‐2, and a Shh‐controlled genetic cascade, including Lmx1a, Msx1 and Nkx6‐1, have been unravelled, acting in parallel or sequentially to establish a territory competent for midbrain dopaminergic precursor production at relatively early stages of neural development. Secondly, the same factors (Wnt1 and Lmx1a/Msx1) appear to regulate midbrain dopaminergic and/or neuronal fate specification in the postmitotic progeny of these precursors by controlling the expression of midbrain dopaminergic‐specific and/or general proneural factors at later stages of neural development. For the first time, early inductive events have thus been linked to later differentiation processes in midbrain dopaminergic neuron development. Given the pivotal importance of this neuronal population for normal function of the human brain and its involvement in severe neurological and psychiatric disorders such as Parkinsons Disease, these advances open new prospects for potential stem cell‐based therapies. We will summarize these new findings in the overall context of midbrain dopaminergic neuron development in this review.

Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon

Meso-diencephalic dopaminergic (mdDA) neurons control voluntary movement, cognition and the reward response, and their degeneration is associated with Parkinsons disease (PD). Prospective cell transplantation therapies for PD require full knowledge of the developmental pathways that control mdDA neurogenesis. We have previously shown that Otx2 is required for the establishment of the mesencephalic field and molecular code of the entire ventral mesencephalon (VM). Here, we investigate whether Otx2 is a specific determinant of mesencephalic dopaminergic (mesDA) neurogenesis by studying mouse mutants that conditionally overexpress or lack Otx2. Our data show that Otx2 overexpression in the VM causes a dose-dependent and selective increase in both mesDA progenitors and neurons, which correlates with a remarkable and specific enhancement in the proliferating activity of mesDA progenitors. Consistently, lack of Otx2 in the VM specifically affects the proliferation of Sox2+ mesDA progenitors and causes their premature post-mitotic transition. Analysis of the developmental pathway that controls the differentiation of mesDA neurons shows that, in the absence of Otx2, the expression of Lmx1a and Msx1, and the proneural genes Ngn2 and Mash1 is not activated in Sox2+ mesDA progenitors, which largely fail to differentiate into Nurr1+ mesDA precursors. Furthermore, proliferation and differentiation abnormalities exhibit increasing severity along the anterior-posterior (AP) axis of the VM. These findings demonstrate that Otx2, through an AP graded effect, is intrinsically required to control proliferation and differentiation of mesDA progenitors. Thus, our data provide new insights into the mechanism of mesDA neuron specification and suggest Otx2 as a potential target for cell replacement-based therapeutic approaches in PD.

Wnt5a Regulates Ventral Midbrain Morphogenesis and the Development of A9–A10 Dopaminergic Cells In Vivo

Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9–10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5–E13.5. Analysis of Wnt5a−/− mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a−/− mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.

Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP.

Mesencephalic-diencephalic dopaminergic neurons control locomotor activity and emotion and are affected in neurodegenerative and psychiatric diseases. The homeoprotein Otx2 is restricted to ventral tegmental area (VTA) neurons that are prevalently complementary to those expressing Girk2 and glycosylated active form of the dopamine transporter (Dat). High levels of glycosylated Dat mark neurons with efficient dopamine uptake and pronounced vulnerability to Parkinsonian degeneration. We found that Otx2 controls neuron subtype identity by antagonizing molecular and functional features of dorsal-lateral VTA, such as Girk2 and Dat expression. Otx2 limited the number of VTA neurons with efficient dopamine uptake and conferred resistance to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Ectopic Otx2 expression also provided neurons of the substantia nigra with efficient neuroprotection to MPTP. These findings indicate that Otx2 is required to specify neuron subtype identity in VTA and may antagonize vulnerability to the Parkinsonian toxin MPTP.

A unilateral negative feedback loop between miR-200 microRNAs and Sox2/E2F3 controls neural progenitor cell-cycle exit and differentiation

MicroRNAs have emerged as key posttranscriptional regulators of gene expression during vertebrate development. We show that the miR-200 family plays a crucial role for the proper generation and survival of ventral neuronal populations in the murine midbrain/hindbrain region, including midbrain dopaminergic neurons, by directly targeting the pluripotency factor Sox2 and the cell-cycle regulator E2F3 in neural stem/progenitor cells. The lack of a negative regulation of Sox2 and E2F3 by miR-200 in conditional Dicer1 mutants (En1+/Cre; Dicer1flox/flox mice) and after miR-200 knockdown in vitro leads to a strongly reduced cell-cycle exit and neuronal differentiation of ventral midbrain/hindbrain (vMH) neural progenitors, whereas the opposite effect is seen after miR-200 overexpression in primary vMH cells. Expression of miR-200 is in turn directly regulated by Sox2 and E2F3, thereby establishing a unilateral negative feedback loop required for the cell-cycle exit and neuronal differentiation of neural stem/progenitor cells. Our findings suggest that the posttranscriptional regulation of Sox2 and E2F3 by miR-200 family members might be a general mechanism to control the transition from a pluripotent/multipotent stem/progenitor cell to a postmitotic and more differentiated cell.

Pitx3 Is a Critical Mediator of GDNF-Induced BDNF Expression in Nigrostriatal Dopaminergic Neurons

Pitx3 is a critical homeodomain transcription factor for the proper development and survival of mesodiencephalic dopaminergic (mdDA) neurons in mammals. Several variants of this gene have been associated with human Parkinsons disease (PD), and lack of Pitx3 in mice causes the preferential loss of substantia nigra pars compacta (SNc) mdDA neurons that are most affected in PD. It is currently unclear how Pitx3 activity promotes the survival of SNc mdDA neurons and which factors act upstream and downstream of Pitx3 in this context. Here we show that a transient expression of glial cell line-derived neurotrophic factor (GDNF) in the murine ventral midbrain (VM) induces transcription of Pitx3 via NF-κB-mediated signaling, and that Pitx3 is in turn required for activating the expression of brain-derived neurotrophic factor (BDNF) in a rostrolateral (SNc) mdDA neuron subpopulation during embryogenesis. The loss of BDNF expression correlates with the increased apoptotic cell death of this mdDA neuronal subpopulation in Pitx3−/− mice, whereas treatment of VM cell cultures with BDNF augments the survival of the Pitx3−/− mdDA neurons. Most importantly, only BDNF but not GDNF protects mdDA neurons against 6-hydroxydopamine-induced cell death in the absence of Pitx3. As the feedforward regulation of GDNF, Pitx3, and BDNF expression also persists in the adult rodent brain, our data suggest that the disruption of the regulatory interaction between these three factors contributes to the loss of mdDA neurons in Pitx3−/− mutant mice and perhaps also in human PD.

Distinct but redundant expression of the Frizzled Wnt receptor genes at signaling centers of the developing mouse brain

The establishment of the regional subdivisions of the vertebrate CNS is accomplished through the activity of different neuroepithelial organizing centers. The wingless/int (Wnt) family of secreted glycoproteins, among other factors, plays a crucial role in signaling from these centers. Wnt1 secreted from the boundary between the mid- and hindbrain, for instance, controls the development of this brain region and of associated neuronal populations. Different Wnts secreted from the caudomedial pallium, the cortical hem, pattern the adjacent hippocampal field. The first step in Wnt signal transduction is binding of the Wnt ligand to its receptors, the seven-pass transmembrane Frizzled proteins. Inactivation of different Frizzled genes in mice have revealed an extensive functional redundancy between these receptors. In order to discriminate between a possible participation of different Frizzled receptors in the transduction of Wnt signals at the mid-/hindbrain boundary and the cortical hem, we have performed a detailed expression study of the 10 known murine Frizzled genes at crucial stages of mouse embryonic development. Our analysis reveals a highly dynamic yet distinct expression pattern of individual Frizzled genes in the anterior neural tube of the developing mouse embryo. The overlapping spatio-temporal expression of at least two and up to six Frizzled genes in any region of the developing mouse brain, however, also suggests a vast functional redundancy of the murine Frizzled receptors. This redundancy has to be taken into consideration for future analyses of Frizzled receptor function at these signaling centers in the mouse.

Nkx6-1 controls the identity and fate of red nucleus and oculomotor neurons in the mouse midbrain

Little is known about the cues controlling the generation of motoneuron populations in the mammalian ventral midbrain. We show that Otx2 provides the crucial anterior-posterior positional information for the generation of red nucleus neurons in the murine midbrain. Moreover, the homeodomain transcription factor Nkx6-1 controls the proper development of the red nucleus and of the oculomotor and trochlear nucleus neurons. Nkx6-1 is expressed in ventral midbrain progenitors and acts as a fate determinant of the Brn3a+ (also known as Pou4f1) red nucleus neurons. These progenitors are partially dorsalized in the absence of Nkx6-1, and a fraction of their postmitotic offspring adopts an alternative cell fate, as revealed by the activation of Dbx1 and Otx2 in these cells. Nkx6-1 is also expressed in postmitotic Isl1+ oculomotor and trochlear neurons. Similar to hindbrain visceral (branchio-) motoneurons, Nkx6-1 controls the proper migration and axon outgrowth of these neurons by regulating the expression of at least three axon guidance/neuronal migration molecules. Based on these findings, we provide additional evidence that the developmental mechanism of the oculomotor and trochlear neurons exhibits more similarity with that of special visceral motoneurons than with that controlling the generation of somatic motoneurons located in the murine caudal hindbrain and spinal cord.

Specification of midbrain territory

The vertebrate neural plate is subdivided into four distinct territories comprising the presumptive forebrain, midbrain, hindbrain, and the spinal cord, shortly after gastrulation. Initially, this subdivision relies on a defined pattern of expression of distinct transcription and secreted factors within the newly formed neuroectoderm, even before morphological partitioning is evident. Subdivision of the neural plate into distinct territories is a complex process, which is also known as patterning or regionalisation, and involves both planar and vertical signals coming from within the neuroectoderm and from neighbouring non-neural tissues. During the course of embryogenesis, this gross subdivision of the neural plate is progressively refined by a variety of mechanisms, leading to the establishment of various subdomains that ultimately give rise to specific cell populations characteristic for the corresponding brain and spinal cord regions. Once again, a prominent feature of these later processes is the defined expression of specific genes within the developing neural tube. In the present review, we will concentrate on the genes active in the progressive refinement of the midbrain territory as a distinct subdivision of the brain. We will also give an outlook on genes that are active during early induction of the anterior neural plate and genetic mechanisms that control the generation of specific cell populations of the ventral midbrain, with special focus on the mesencephalic dopaminergic neurons.