Dll1 haploinsufficiency causes brain abnormalities with functional relevance.

Introduction: The Notch pathway is fundamental for the generation of neurons during development. We previously reported that adult mice heterozygous for the null allele of the gene encoding the Delta-like ligand 1 for Notch (Dll1lacZ ) have a reduced neuronal density in the substantia nigra pars compacta. The aim of the present work was to evaluate whether this alteration extends to other brain structures and the behavioral consequences of affected subjects. Methods: Brains of Dll1 +/lacZ embryos and mice at different ages were phenotypically compared against their wild type (WT) counterpart. Afterwards, brain histological analyses were performed followed by determinations of neural cell markers in tissue slices. Neurological deficits were diagnosed by applying different behavioral tests to Dll1 +/lacZ and WT mice. Results: Brain weight and size of Dll1 +/lacZ mice was significantly decreased compared with WT littermates (i.e., microcephaly), a phenotype detected early after birth. Interestingly, enlarged ventricles (i.e., hydrocephalus) was a common characteristic of brains of Dll1 haploinsufficient mice since early ages. At the cell level, general cell density and number of neurons in several brain regions, including the cortex and hippocampus, of Dll1 +/lacZ mice were reduced as compared with those regions of WT mice. Also, fewer neural stem cells were particularly found in the adult dentate gyrus of Dll1 +/lacZ mice but not in the subventricular zone. High myelination levels detected at early postnatal ages (P7-P24) were an additional penetrant phenotype in Dll1 +/lacZ mice, observation that was consistent with premature oligodendrocyte differentiation. After applying a set of behavioral tests, mild neurological alterations were detected that caused changes in motor behaviors and a deficit in object categorization. Discussion: Our observations suggest that Dll1 haploinsufficiency limits Notch signaling during brain development which, on one hand, leads to reduced brain cell density and causes microcephaly and hydrocephalus phenotypes and, on the other, alters the myelination process after birth. The severity of these defects could reach levels that affect normal brain function. Therefore, Dll1 haploinsufficiency is a risk factor that predisposes the brain to develop abnormalities with functional consequences.

The Substantia Nigra Is Permissive and Gains Inductive Signals When Lesioned for Dopaminergic Differentiation of Embryonic Stem Cells.

Transplantation of dopaminergic (DA) cells into the striatum can rescue from dopamine deficiency in a Parkinson's disease condition, but this is not a suitable procedure for regaining the full control of motor activity. The minimal condition toward recovering the nigrostriatal pathway is the proper innervation of transplanted DA neurons or their precursors from the substancia nigra pars compacta (SNpc) to their target areas. However, functional integration of transplanted cells would require first that the host SNpc is suitable for their survival and/or differentiation. We recently reported that the intact adult SNpc holds a strong neurogenic environment, but primed embryonic stem cells (ie, embryoid body cells, EBCs) could not derive into DA neurons. In this study, we transplanted into the intact or lesioned SNpc, EBCs derived from embryonic stem cells that were prompt to differentiate into DA neurons by the forced expression of Lmx1a in neural precursor cells (R1B5/NesE-Lmx1a). We observed that, 6 days posttransplantation (dpt), R1B5 or R1B5/NesE-Lmx1a EBCs gave rise to Nes+ and Dcx+ cells within the host SNpc, but a large number of Th+ cells derived only from EBCs exogenously expressing Lmx1a. In contrast, when transplantation was carried out into the 6-hydroxidopamine-lesioned SNpc, the emergence of Th+ cells from EBCs was independent of exogenous Lmx1a expression, although these cells were not found by 15 dpt. These results suggest that the adult SNpc is not only a permissive niche for initiation of DA differentiation of non-neuralized cells but also releases factors upon damage that promote the acquisition of DA characteristics by transplanted EBCs.

Functional determination of the differentiation potential of ventral mesencephalic neural precursor cells during dopaminergic neurogenesis.

The ventral mesencephalic neural precursor cells (vmNPCs) that give rise to dopaminergic (DA) neurons have been identified by the expression of distinct genes (e.g., Lmx1a, Foxa2, Msx1/2). However, the commitment of these NPCs to the mesencephalic DA neuronal fate has not been functionally determined. Evaluation of the plasticity of vmNPCs suggests that their commitment occurs after E10.5. Here we show that E9.5 vmNPCs implanted in an ectopic area of E10.5 mesencephalic explants, retained their specification marker Lmx1a and efficiently differentiated into neurons but did not express the gene encoding tyrosine hydroxylase (Th), the limiting enzyme for dopamine synthesis. A proportion of E10.5-E11.5 implanted vmNPCs behaved as committed, deriving into Th+ neurons in ectopic sites. Interestingly, implanted cells from E12.5 embryos were unable to give rise to a significant number of Th+ neurons. Concomitantly, differentiation assays in culture and in mesencephalic explants treated with Fgf2+LIF detected vmNPCs with astrogenic potential since E11.5. Despite this, a full suspension of E12.5 vmNPCs give rise to DA neurons in a similar proportion as those of E10.5 when they were transplanted into adult brain, but astrocytes were only detected with the former population. These data suggest that the subventricular postmitotic progenitors present in E12.5 ventral mesencephalon are unable to implant in embryonic explants and are the source of DA neurons in the transplanted adult brain. Based on our findings we propose that during DA differentiation committed vmNPCs emerge at E10.5 and they exhaust their neurogenic capacity with the rise of NPCs with astrogenic potential.

Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain.

Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs), but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+). These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons.

Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

Telencephalic neural precursor cells show transient competence to interpret the dopaminergic niche of the embryonic midbrain.

Neural Precursor Cells (NPCs) generate complex stereotypic arrays of neuronal subtypes in the brain. This process involves the integration of patterning cues that progressively restrict the fate of specific NPCs. Yet the capacity of NPCs to interpret foreign microenvironments during development remains poorly defined. The aim of this work was to test the competence of mouse telencephalic NPCs to respond to the dopaminergic niche of the mesencephalon. Telencephalic NPCs isolated from midgestation mouse embryos (E10.5) and transplanted to age-matched mesencephalic explants efficiently differentiated into neurons but were largely unable to produce midbrain dopaminergic (mDA) neurons. Instead, E10.5 telencephalic NPCs behaved as restricted gabaergic progenitors that maintained ectopic expression of Foxg1 and Pax6. In contrast, E8.5 telencephalic NPCs were able to differentiate into Lmx1a(+)/Foxa2(+)/TH(+) neurons in the dopaminergic niche of the mesencephalic explants. In addition, these early telencephalic NPCs showed region-dependent expression of Nkx6.1, Nkx2.2 and site-specific differentiation into gabaergic neurons within the mesencephalic tissue. Significant dopaminergic differentiation of E8.5 telencephalic NPCs was not observed after transplantation to E12.5 mesencephalic explants, suggesting that inductive signals in the dopaminergic niche rapidly decay after midgestation. Moreover, we employed transplantation of embryonic stem cells-derived precursors to demonstrate that extinction of inductive signals within the telencephalon lags behind the commitment of residing NPCs. Our data indicate that the plasticity to interpret multiple instructive niches is an early and ephemeral feature of the telencephalic neural lineage.