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Sagittal sections of E12.5, E13.5 and E14.5 wild-type mouse brains, from lateral to medial to lateral. ISH staining is shown for Phox2b, and for Lmx1b as a reference. Dashed lines represent the mid-hindbrain boundary. (a–l) Lmx1b and Phox2b mRNA expression at E12.5. Lmx1b is broadly expressed throughout the midbrain, in P1, P2 and P3, and in the hindbrain. Except for the most medial part, Phox2b is expressed in the posterior midbrain and anterior hindbrain. (m–x) Lmx1b and Phox2b mRNA expression at E13.5, and (y–jj) at E14.5. (kk) Schematic overview of a sagittal mouse brain at E14.5, depicting several neuronal fields in the midbrain area (red box). C, caudal; R, rostral; FB, forebrain; MB, midbrain; HB, hindbrain; RN, red nucleus; OM, oculomotor complex.
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The developing mesodiencephalic dopaminergic (mdDA) neuronal field can be subdivided into several molecularly distinct domains that arise due to spatiotemporally distinct origins of the neurons and distinct transcriptional pathways controlling these neuronal subsets. Two large anatomically and functionally different subdomains are formed that event...
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... For instance, in control HCOs, some PHOX2B + cells were found in the OTX2 + domain of the mid-hindbrain boundary (MHB), but this was not observed in mutant HCOs ( Figure 6J), implying that the genetic program for the positioning of PHOX2B + cells may be altered in mutant HCOs. Phox2b has been suggested to regulate dopaminergic neuron development in the embryonic mouse midbrain (Hoekstra et al., 2012), so PHOX2B-7Ala may also interrupt the formation of dopaminergic neurons in the MHB. Taken together, we found that the pattern specification genes were severely interrupted in the mutant PHOX2B + cells, with a concomitant loss of PHOX2B + cells in the respiratory center and OTX2 + domain in the PHOX2B-7Ala HCOs. ...
Retrotrapezoid nucleus (RTN) neurons in the brainstem regulate the ventilatory response to hypercarbia. It is unclear how PHOX2B-polyalanine repeat mutations (PHOX2B-PARMs) alter the function of PHOX2B and perturb the formation of RTN neurons. Here, we generated human brainstem organoids (HBSOs) with RTN-like neurons from human pluripotent stem cells. Single-cell transcriptomics revealed that expression of PHOX2B+7Ala PARM alters the differentiation trajectories of the hindbrain neurons and hampers the formation of the RTN-like neurons in HBSOs. With the unguided cerebral organoids (HCOs), PHOX2B+7Ala PARM interrupted the patterning of PHOX2B+ neurons with dysregulation of Hedgehog pathway and HOX genes. With complementary use of HBSOs and HCOs with a patient and two mutant induced pluripotent stem cell lines carrying different polyalanine repetition in PHOX2B, we further defined the association between the length of polyalanine repetition and malformation of RTN-respiratory center and demonstrated the potential toxic gain of function of PHOX2B-PARMs, highlighting the uniqueness of these organoid models for disease modeling.
... This could indicate a role for Bcl11a in the differentiation of mDA precursors into a Bcl11a + subtype 40 . In addition to the aforementioned transcription factors in this section, additional genes encoding transcription factors and other proteins have been implicated in the early development of mouse mDA neuron subsets: Dcc (SNc) 111 , Phox2b (dorsocaudal VTA) 112 , Tcf4 (VTA and Aldh1a1 + subsets) 113 , Ebf1 (which encodes early B cell factor 1; SNc) 114 and Uncx4.1 (also known as Uncx, which encodes UNC homeobox 4.1; Calb1 + subset and SNpl) 115 . Although further studies are needed to examine the role of these cues in more detail, it is becoming evident that the combinatorial actions of different intrinsic (and probably extrinsic) molecular regulators is required to dictate the birth and subsequent differentiation of mDA neuron subtypes. ...
The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviours and are linked to various brain diseases. Considerable progress has been made in identifying mDA neuron subtypes, and recent work has begun to unveil how these neuronal subtypes develop and organize into functional brain structures. This progress is important for further understanding the disparate physiological functions of mDA neurons and their selective vulnerability in disease, and will ultimately accelerate therapy development. This Review discusses recent advances in our understanding of molecularly defined mDA neuron subtypes and their circuits, ranging from early developmental events, such as neuron migration and axon guidance, to their wiring and function, and future implications for therapeutic strategies. Recent technological advances have provided insights into the diversity of neuronal subtypes within the midbrain dopamine system. In this Review, Garritsen and colleagues discuss molecular and functional distinctions between subtypes and describe mechanisms underlying their development, wiring and function.
... Large rodent-based micro-array studies determined that the difference in molecular profile between the SNc and VTA is smaller than 3% [3][4][5]; reviewed in [6]. Several studies from our group contributed to the quest to define molecular profiles for different mdDA subsets already during embryonic development [7][8][9][10]. Recently, elegant transcriptomic studies identified these molecular profiles in single-cellular resolution in early post-natal tissue [11], as well as during embryonic development of both murine and human tissue [12]. ...
Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral midbrain. These neurons form the substantia nigra (SNc) and the ventral tegmental area (VTA). Two transcription factors that play important roles in the process of terminal differentiation and subset-specification of mdDA neurons, are paired-like homeodomain transcription factor 3 (Pitx3), and homeobox transcription factor Engrailed 1 (En1). We previously investigated the single Pitx3KO and En1KO and observed important changes in the survival of mdDA neurons of the SNc and VTA as well as altered expression of pivotal rostral- and caudal-markers, Ahd2 and Cck, respectively. To refine our understanding of the regional-specific relationships between En1 and Pitx3 and their (combined) role in the programming mdDA neurons on the rostral-to-caudal axis, we created double En1tm1Alj/tm1Alj;Pitx3gfp/gfp (En1KO; Pitx3GFP/GFP) animals. Here we report, that in absence of En1 and Pitx3, only a limited number of mdDA neurons are present at E14.5. These mdDA neurons have a rudimentary dopaminergic cell fate, as they express Nurr1, Pbx3 and Otx2 but have lost their rostral or caudal subset identity. Furthermore, we report that the expression of Cck depends on En1 expression, while (in contrast) both Pitx3 and En1 are involved in the initiation of Ahd2 expression. Thus we reveal in this manuscript that regulated levels of Pitx3 and En1 control the size and rostral/caudal-identity of the mdDA neuronal population. © 2017 Kouwenhoven et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... In most of the brainstem, Phox2b represses serotonergic differentiation and therefore it is required to be absent for specification of raphe serotonergic neurons. Although Phox2b is expressed in caudal midbrain dopaminergic populations, it does not seem to be required for the specification of substantia nigra, pars compacta (SNC) DN. 214 In mice, Lmx1b expression is required for the expression of monoamine vesicular transporters in all brainstem aminergic neurons (dopaminergic, noradrenergic and serotonergic), 51,109,130,174 despite not being required for zebrafish LC noradrenergic populations. 184 Visceromotor neurons of the 10N do not express vesicular monoamine transporters, 33 but it is not known if Lmx1b is required for the development of these neurons. ...
A specific set of brainstem nuclei are susceptible to degeneration in Parkinson’s disease. We hypothesise that neuronal vulnerability reflects shared phenotypic characteristics that confer selective vulnerability to degeneration. Neuronal phenotypic specification is mainly the cumulative result of a transcriptional regulatory program that is active during the development. By manual curation of the developmental biology literature, we comprehensively reconstructed an anatomically resolved cellular developmental lineage for the adult neurons in five brainstem regions that are selectively vulnerable to degeneration in prodromal or early Parkinson’s disease. We synthesised the literature on transcription factors that are required to be active, or required to be inactive, in the development of each of these five brainstem regions, and at least two differentially vulnerable nuclei within each region. Certain transcription factors, e.g., Ascl1 and Lmx1b, seem to be required for specification of many brainstem regions that are susceptible to degeneration in early Parkinson’s disease. Some transcription factors can even distinguish between differentially vulnerable nuclei within the same brain region, e.g., Pitx3 is required for specification of the substantia nigra pars compacta, but not the ventral tegmental area. We do not suggest that Parkinson’s disease is a developmental disorder. In contrast, we consider identification of shared developmental trajectories as part of a broader effort to identify the molecular mechanisms that underlie the phenotypic features that are shared by selectively vulnerable neurons. Systematic in vivo assessment of fate determining transcription factors should be completed for all neuronal populations vulnerable to degeneration in early Parkinson’s disease.
... In situ hybridization was performed as described previously (Smits et al., 2003). Digoxigenin-labeled probes for Th, En2, Vmat2, Dat, Nurr1, AADC, Lmx1b, Pitx3, Pbx1, Pbx3 and Wnt1 were used as previously described (Grima et al., 1985;Hoekstra et al., 2012;Mesman et al., 2014;Smidt et al., 2000a,b;Veenvliet et al., 2013). ...
The isthmic organizer (IsO) is a signaling center that specifies the correct and distinct embryonic development of the dopaminergic midbrain and serotonergic hindbrain. The IsO is a linear boundary between the two brain regions, emerging at around embryonic day 7-8 of murine embryonic development, that shapes its surroundings through the expression of instructive signals such as Wnt and growth factors. Homeobox transcription factor engrailed 1 (En1) is present in midbrain and rostral hindbrain (i.e. rhombomere 1, R1). Its expression spans the IsO, and it is known to be an important survival factor for both dopaminergic and serotonergic neurons. Erroneous composition of dopaminergic neurons in the midbrain or serotonergic neurons in the hindbrain is associated with severe pathologies such as Parkinson's disease, depression or autism. Here we investigated the role of En1 in early mid-hindbrain development, using multiple En1-ablated mouse models as well as lineage-tracing techniques, and observed the appearance of ectopic dopaminergic neurons, indistinguishable from midbrain dopaminergic neurons based on molecular profile and intrinsic electrophysiological properties. We propose that this change is the direct result of a caudal relocation of the IsO as represented by ectopic presence of Fgf8, Otx2, Wnt1 and canonical Wnt-signalling. Our work suggests a newly-discovered role for En1: the repression of Otx2, Wnt1 and canonical Wnt-signaling in R1. Overall, our results suggest that En1 is essential for proper IsO maintenance and function.
... The unique expression pattern of Phox2b has been reported in mice, zebrafish, and lamprey during embryonic development [29,30,[48][49][50][51], and in adult rat brain [13,17]. Furthermore, the clinical symptoms of disease caused by mutations in the coding sequence of human PHOX2B are consistent with the expression patterns observed in rodents [31]. ...
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.
Subtle mood fluctuations are normal emotional experiences, whereas drastic mood swings can be a manifestation of bipolar disorder (BPD). Despite their importance for normal and pathological behavior, the mechanisms underlying endogenous mood instability are largely unknown. During embryogenesis, the transcription factor Otx2 orchestrates the genetic networks directing the specification of dopaminergic (DA) and serotonergic (5-HT) neurons. Here we behaviorally phenotyped mouse mutants overexpressing Otx2 in the hindbrain, resulting in an increased number of DA neurons and a decreased number of 5-HT neurons in both developing and mature animals. Over the course of 1 month, control animals exhibited stable locomotor activity in their home cages, whereas mutants showed extended periods of elevated or decreased activity relative to their individual average. Additional behavioral paradigms, testing for manic-and depressive-like behavior, demonstrated that mutants showed an increase in intra-individual fluctuations in locomotor activity, habituation, risk-taking behavioral parameters, social interaction, and hedonic-like behavior. Olanzapine, lithium, and carbamazepine ameliorated the behavioral alterations of the mutants, as did the mixed serotonin receptor agonist quipazine and the specific 5-HT2C receptor agonist CP-809101. Testing the relevance of the genetic networks specifying monoaminergic neurons for BPD in humans, we applied an interval-based enrichment analysis tool for genome-wide association studies. We observed that the genes specifying DA and 5-HT neurons exhibit a significant level of aggregated association with BPD but not with schizophrenia or major depressive disorder. The results of our translational study suggest that aberrant development of monoaminergic neurons leads to mood fluctuations and may be associated with BPD.
The populations of most nations consume products of both domestic and foreign origin, importing together with the products the water which is expended abroad for their production (termed 'virtual water'). Therefore, any investigation of the sustainability of present-day water consumption under future climate change needs to consider the effects of potentially reduced water availability both on domestic water resources and on the trades of virtual water. Here we use combinations of Global Climate and Global Impact Models from the ISI–MIP ensemble to derive patterns of future water availability under the RCP2.6 and RCP8.5 greenhouse gas (GHG) concentrations scenarios. We assess the effects of reduced water availability in these scenarios on national water consumptions and virtual water trades through a simple accounting scheme based on the water footprint concept. We thereby identify countries where the water footprint within the country area is reduced due to a reduced within-area water availability, most prominently in the Mediterranean and some African countries. National water consumption in countries such as Russia, which are non-water scarce by themselves, can be affected through reduced imports from water scarce countries. We find overall stronger effects of the higher GHG concentrations scenario, although the model range of climate projections for single GHG concentrations scenarios is in itself larger than the differences induced by the GHG concentrations scenarios. Our results highlight that, for both investigated GHG concentration scenarios, the current water consumption and virtual water trades cannot be sustained into the future due to the projected patterns of reduced water availability.
This work quantifies the agricultural water footprint (WF) of production (WFprod, agr) and consumption (WFcons, agr) and the resulting net virtual water import (netVWi, agr) of 365 European river basins for a reference period (REF, 1996–2005) and two diet scenarios (a healthy diet based upon food-based dietary guidelines (HEALTHY) and a vegetarian (VEG) diet). In addition to total (tot) amounts, a differentiation is also made between the green (gn), blue (bl) and grey (gy) components. River basins where the REF WFcons, agr, tot exceeds the WFprod, agr, tot (resulting in positive netVWi, agr, tot values), are found along the London–Milan axis. These include the Thames, Scheldt, Meuse, Seine, Rhine and Po basins. River basins where the WFprod, agr, tot exceeds the WFcons, agr, tot are found in Western France, the Iberian Peninsula and the Baltic region. These include the Loire, Ebro and Nemunas basins. Under the HEALTHY diet scenario, the WFcons, agr, tot of most river basins decreases (max −32%), although it was found to increase in some basins in northern and eastern Europe. This results in 22 river basins, including the Danube, shifting from being net VW importers to being net VW exporters. A reduction (max −46%) in WFcons, agr, tot is observed for all but one river basin under the VEG diet scenario. In total, 50 river basins shift from being net VW importers to being net exporters, including the Danube, Seine, Rhone and Elbe basins. Similar observations are made when only the gn + bl and gn components are assessed. When analysing only the bl component, a different river basin pattern is observed.
