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Transcriptome profiling of the WNT/b-catenin-responding mdDA domain in the E12.5 BAT-gal⁺ mouse VM. (A) Representative coronal section at medial midbrain level of an E12.5 BAT-gal⁺ embryo, immunostained with antibodies against b-GAL (green), and PITX3 (red). Red broken line delimits the WNT/b-catenin-responding mdDA domain, orange broken line delimits the lateral non-WNT/b-catenin-responding non-mdDA domain, which were laser-microdissected and used for the comparative microarray-based transcriptome analysis. Right side depicts the legend for the heat map analysis of differentially regulated genes (FDR < 10%) in the laser-microdissected (LMD) WNT/b-catenin-responding mdDA domain (mdDA) relative to the lateral non-WNT/b-catenin-responding non-mdDA domain (Lateral). Data of FACS-sorted GFP-expressing mdDA neurons from E13.5 and E14.5 Pitx3GFP/+ embryos (E13.5, E14.5) are shown in addition. Two technical replicates were analyzed for each sample. (B,C) Heat maps representing expression levels (yellow: overrepresented; blue: underrepresented) of selected mdDA neuron-associated (B) or WNT signaling-associated (C) genes in the laser-microdissected WNT/b-catenin-responding mdDA domain relative to the lateral non-WNT/b-catenin-responding non-mdDA domain and FACS-sorted E13.5/E14.5 Pitx3GFP/+ mdDA neurons. Average fold-changes shown are WNT/b-catenin-responding mdDA domain vs. lateral non-WNT/b-catenin-responding non-mdDA domain. Yellow and blue boxes denote the genes whose expression patterns were analyzed in situ. (D–J”) Representative cresyl violet-stained coronal sections (dorsal top) at different rostrocaudal levels of the BAT-gal⁺ midbrain at E12.5, hybridized with riboprobes for Fgf14 (D–D”), Cck (E–E”), Sulf1 (F–F”), Smarca1 (G–G”), Apcdd1 (H–H”), Pbx1 (I–I”), and Sulf2 (J–J”). (D–E”,G–G”), dark-field images. (K–K”) Representative close-up views of the VM on coronal sections (dorsal top) at different rostrocaudal levels of the BAT-gal⁺ midbrain at E10.5, immunostained for b-GAL (green). Broken white lines delimit the FP from BP. Scale bars: 200 μm (A,D); 50 μm (K”).
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The mesodiencephalic dopaminergic (mdDA) neurons, including the nigrostriatal subset that preferentially degenerates in Parkinson’s Disease (PD), strongly depend on an accurately balanced Wingless-type MMTV integration site family member 1 (WNT1)/beta-catenin signaling pathway during their development. Loss of this pathway abolishes the generation...
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... Wnt signalling plays a decisive role in the initial stage of hair growth. 10,30 β-Catenin, [31][32][33][34] a key regulatory protein of the classic Wnt signalling pathway, regulates hair growth by activating the nuclear auxiliary transcription factor LEF1, 35 Previous reports have demonstrated that Wnt/β-catenin signalling plays an important role in skin homeostasis, and our results here also demonstrate that MLT activates Wnt/β-catenin signalling during hair regeneration. However, the types of cells that are activated by MLT in the Wnt/β-catenin signalling pathway remain unclear. ...
Melatonin (MLT) is a circadian hormone that reportedly influences the development and cyclic growth of secondary hair follicles; however, the mechanism of regulation remains unknown. Here, we systematically investigated the role of MLT in hair regeneration using a hair depilation mouse model. We found that MLT supplementation significantly promoted hair regeneration in the hair depilation mouse model, whereas supplementation of MLT receptor antagonist luzindole significantly suppressed hair regeneration. By analysing gene expression dynamics between the MLT group and luzindole‐treated groups, we revealed that MLT supplementation significantly up‐regulated Wnt/β‐catenin signalling pathway‐related genes. In‐depth analysis of the expression of key molecules in the Wnt/β‐catenin signalling pathway revealed that MLT up‐regulated the Wnt/β‐catenin signalling pathway in dermal papillae (DP), whereas these effects were facilitated through mediating Wnt ligand expression levels in the hair follicle stem cells (HFSCs). Using a DP‐HFSCs co‐culture system, we verified that MLT activated Wnt/β‐catenin signalling in DPs when co‐cultured with HFSCs, whereas supplementation of DP cells with MLT alone failed to activate Wnt/β‐catenin signalling. In summary, our work identified a critical role for MLT in promoting hair regeneration and will have potential implications for future hair loss treatment in humans.
... Thus, WNT1 deficiency is likely to occur only in these portions of cells. Secondly, a report conducted on BAT-gal reporter mice found that (data showed in its Supplemental materials) the number of DAergic progenitor cells in response to ß-catenin (Pitx3 + /ß-GAL + ) was decreased following Wnt1 knockout, but the number of these cells (Pitx3 + /ß-GAL + ) accounts only for a very low proportion of the total number of ß-GAL + cells [29]. Therefore, Western blot with bulk embryonic brain tissue may not be sufficient to detect such minor changes; (2) There may be a compensatory activation of WNT/ß-catenin signaling pathway. ...
Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders with a strong genetic liability. Despite extensive studies, however, the underlying pathogenic mechanism still remains elusive. In the present study, we identified a homozygous mutation in the intron 1 of Wnt1 via large-scale screening of ASD risk/causative genes and verified that this mutation created a new splicing donor site in the intron 1, and consequently, a decrease of WNT1 expression. Interestingly, humanized rat models harboring this mutation exhibited robust ASD-like behaviors including impaired ultrasonic vocalization (USV), decreased social interactions, and restricted and repetitive behaviors. Moreover, in the substantia nigra compacta (SNpc) and the ventral tegmental area (VTA) of mutant rats, dopaminergic (DAergic) neurons were dramatically lost, together with a comparable decrease in striatal DAergic fibers. Furthermore, using single-cell RNA sequencing, we demonstrated that the decreased DAergic neurons in these midbrain areas might attribute to a shift of the boundary of the local pool of progenitor cells from the hypothalamic floor plate to the midbrain floor plate during the early embryonic stage. Moreover, treatments of mutant rats with levodopa could attenuate the impaired USV and social interactions almost completely, but not the restricted and repetitive behaviors. Our results for the first time documented that the developmental loss of DAergic neurons in the midbrain underlies the pathogenesis of ASD, and that the abnormal progenitor cell patterning is a cellular underpinning for this developmental DAergic neuronal loss. Importantly, the effective dopamine therapy suggests a translational significance in the treatment of ASD.
... Wnt1/β-catenin/LEF1 signaling plays an essential role in the development of the mouse's ventral midbrain [50,51]. Lmx1a and Pitx3 are all potential direct targets of Wnt1/βcatenin/LEF1-mediated signaling [52], suggesting a cascade linking Wnt1/β-catenin/LEF1-Lmx1a-Pitx3 to mDA differentiation. Moreover, Lmx1b is also closely associated with Wnt1 signaling pathway, and the Wnt1-Lmx1b autoregulatory loop directly regulates Nurr1, En1, and Pitx3, all of which are crucial elements in mDA neuronal differentiation and survival [53,54]. ...
The degeneration of midbrain dopaminergic (mDA) neurons, particularly in the substantia nigra pars compacta (SNc), is one of the most prominent pathological hallmarks of Parkinson’s disease (PD). To uncover the pathogenic mechanisms of mDA neuronal death during PD may provide therapeutic targets to prevent mDA neuronal loss and slow down the disease’s progression. Paired-like homeodomain transcription factor 3 (Pitx3) is selectively expressed in the mDA neurons as early as embryonic day 11.5 and plays a critical role in mDA neuron terminal differentiation and subset specification. Moreover, Pitx3-deficient mice exhibit some canonical PD-related features, including the profound loss of SNc mDA neurons, a dramatic decrease in striatal dopamine (DA) levels, and motor abnormalities. However, the precise role of Pitx3 in progressive PD and how this gene contributes to mDA neuronal specification during early stages remains unclear. In this review, we updated the latest findings on Pitx3 by summarizing the crosstalk between Pitx3 and its associated transcription factors in mDA neuron development. We further explored the potential benefits of Pitx3 as a therapeutic target for PD in the future. To better understand the transcriptional network of Pitx3 in mDA neuron development may provide insights into Pitx3-related clinical drug-targeting research and therapeutic approaches.
... Alterations in Wnt signalling strength contribute to mDA neuron subtype specification 90,93,94 (Fig. 2a). Various Wnt signalling molecules are present in the caudolateral floorplate 95 . In the lateral floorplate, R-spondin 2 (RSPO2) enhances Wnt signalling [95][96][97] , which activates lymphoid enhancer-binding factor 1 (LEF1). ...
... Various Wnt signalling molecules are present in the caudolateral floorplate 95 . In the lateral floorplate, R-spondin 2 (RSPO2) enhances Wnt signalling [95][96][97] , which activates lymphoid enhancer-binding factor 1 (LEF1). LEF1 induces Lmx1a 98 , but hampers LMX1A in activating Pitx3. ...
... 99 ). DKK3 reduces LEF1 expression by downregulating Wnt signalling, allowing Pitx3 expression through LMX1A and pre-B cell leukaemia transcription factor 1 (PBX1) 95,99 . Interestingly, the rostrolateral SNc is affected when Lmx1a is ablated 97 . ...
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.
... 161 The development of mesodiencephalic dopaminergic (mdDA) neurons and mdDA progenitors, including the nigrostriatal subset that preferentially degenerates in PD, depends on Wnt/β-catenin signalling. 162 Ghrelin, an endogenous ligand for growth hormone secretagogue receptor 1a, plays a fundamental role in regulating energy homeostasis in PD. Wnt/β-catenin signalling is relevant to DAergic neuron differentiation induced by ghrelin. ...
The Wnt/β-catenin pathway comprises a family of proteins that play critical roles in embryonic development and adult tissue homeostasis. The deregulation of Wnt/β-catenin signalling often leads to various serious diseases, including cancer and non-cancer diseases. Although many articles have reviewed Wnt/β-catenin from various aspects, a systematic review encompassing the origin, composition, function, and clinical trials of the Wnt/β-catenin signalling pathway in tumour and diseases is lacking. In this article, we comprehensively review the Wnt/β-catenin pathway from the above five aspects in combination with the latest research. Finally, we propose challenges and opportunities for the development of small-molecular compounds targeting the Wnt signalling pathway in disease treatment.
... We similarly scan each nucleotide in the CNE using a window size of ~10bp and find strong acceleration for a segment that corresponds to the binding site for LMX1A (LIM homeobox transcription factor 1 alpha), a midbrainspecific transcription factor (Uhde et al. 2010). WNT1 and LMX1A regulate the midbrain dopaminergic neuronal differentiation via an autoregulatory loop (Chung et al. 2009), which is also mediated by LEF1 (Nouri et al. 2020). The promoter of LMX1A contains a binding site for LEF1/T-cell factor (Nouri et al. 2020), but the presence of the strongly accelerated binding site for LMX1A in this CNE suggests that a possible feedforward mechanism may be involved in maintaining the levels of LMX1A and LEF1 during development. ...
... WNT1 and LMX1A regulate the midbrain dopaminergic neuronal differentiation via an autoregulatory loop (Chung et al. 2009), which is also mediated by LEF1 (Nouri et al. 2020). The promoter of LMX1A contains a binding site for LEF1/T-cell factor (Nouri et al. 2020), but the presence of the strongly accelerated binding site for LMX1A in this CNE suggests that a possible feedforward mechanism may be involved in maintaining the levels of LMX1A and LEF1 during development. All in all, these examples demonstrate that phyloConverge is able to highlight portions of an element that are potentially important for the convergent phenotype of interest and that then lead to hypotheses that are amenable to experimental interrogation. ...
Physiological and morphological adaptations to extreme environments arise from the molecular evolution of protein-coding regions and regulatory elements (REs) that regulate gene expression. Comparative genomics methods can characterize genetic elements that underlie the organism-level adaptations, but convergence analyses of REs are often limited by their evolutionary properties. A RE can be modularly composed of multiple transcription factor binding sites (TFBS) that may each experience different evolutionary pressures. The modular composition and rapid turnover of TFBS also enables a compensatory mechanism among nearby TFBS that allows for weaker sequence conservation/divergence than intuitively expected. Here, we introduce phyloConverge, a comparative genomics method that can perform fast, fine-grained local convergence analysis of genetic elements. phyloConverge calibrates for local shifts in evolutionary rates using a combination of maximum likelihood-based estimation of nucleotide substitution rates and phylogenetic permutation tests. Using the classical convergence case of mammalian adaptation to subterranean environments, we validate that phyloConverge identifies rate-accelerated conserved non-coding elements (CNEs) that are strongly correlated with ocular tissues, with improved specificity compared to competing methods. We use phyloConverge to perform TFBS-scale and nucleotide-scale scoring to dissect each CNE into subregions with uneven convergence signals and demonstrate its utility for understanding the modularity and pleiotropy of REs. Subterranean-accelerated regions are also enriched for molecular pathways and TFBS motifs associated with neuronal phenotypes, suggesting that subterranean eye degeneration may coincide with a remodeling of the nervous system. phyloConverge offers a rapid and accurate approach for understanding the evolution and modularity of regulatory elements underlying phenotypic adaptation.
... In fact, it was recently shown that inhibition of WNT signaling during the late phase of VM/mDA NSC/NPC patterning (after initially higher WNT signaling levels compared to our protocol [26]) promotes the preferential derivation of SNc DA neurons from human PSCs, whereas high WNT signaling levels during this phase force them into the VTA DA neuron identity [36]. We therefore speculate that an improved protocol, which precisely recapitulates the time course of WNT signaling during mDA neuron development in vivo [12,37] might be able to direct our NES-mScarlet hiPSCs into a genuine and possibly also subtype-specific mDA neuron identity. This will be assessed in future experiments. ...
Advances in the regenerative stem cell field have propelled the generation of tissue-specific cells in the culture dish for subsequent transplantation, drug screening purposes, or the elucidation of disease mechanisms. One major obstacle is the heterogeneity of these cultures, in which the tissue-specific cells of interest usually represent only a fraction of all generated cells. Direct identification of the cells of interest and the ability to specifically isolate these cells in vitro is, thus, highly desirable for these applications. The type VI intermediate filament protein NESTIN is widely used as a marker for neural stem/progenitor cells (NSCs/NPCs) in the developing and adult central and peripheral nervous systems. Applying CRISPR-Cas9 technology, we have introduced a red fluorescent reporter (mScarlet) into the NESTIN (NES) locus of a human induced pluripotent stem cell (hiPSC) line. We describe the generation and characterization of NES-mScarlet reporter hiPSCs and demonstrate that this line is an accurate reporter of NSCs/NPCs during their directed differentiation into human midbrain dopaminergic (mDA) neurons. Furthermore, NES-mScarlet hiPSCs can be used for direct identification during live cell imaging and for flow cytometric analysis and sorting of red fluorescent NSCs/NPCs in this paradigm.
... As the IsO is already long established after E14.5, we set out to determine whether early patterning of the IsO is affected upon loss of Nkx2.9. To this end, we examined the expression of Wnt1, an important regulator of the IsO and midbrain area [32][33][34], and the expression of Fgf8, the quintessential marker of the IsO [35,36] in the Nkx2.9-ablated midbrain at E12.5. ...
... Taken together, these data further suggest that initial subset programming might be influenced by Nkx2.9. However, as expression of Wnt8b is affected in the Nkx2.9 mutant, we cannot rule out the possibility that the effects on Dat and Cck expression at E14.5 may be caused by affected WNT-signaling in this area, considering that a subset of Dat-expressing cells is responsive for canonical Wnt-signaling [32]. Interestingly, in zebrafish, it has been shown that Wnt8b is involved in establishment of the IsO, and loss of Wnt8b results in an aberrant number of dopamine neurons in the zebrafish diencephalon [25,27]. ...
Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.
... Similarly, we observed pitx2, lef1, and tcf7l2 specifically in the midbrain. These wnt-mediating genes have also been observed primarily in mesodiencephalic DOP neurons (Martin et al. 2004;Nouri et al. 2020), consistent with our observation of DOP neuron marker enrichment in the midbrain ( Figure 3E). Thus, pathway enrichment in the regional comparisons also appear to reflect regional differences rather than any significant disparities in proliferation or differentiation. ...
Amphibian metamorphosis is a transitional period that involves significant changes in the cell type populations and biological processes occurring in the brain. Analysis of gene expression dynamics during this process may provide insight into the molecular events underlying these changes. We conducted differential gene expression analyses of the developing X. laevis tadpole brain during this period in two ways: first, over stages of development in the midbrain, and second, across regions of the brain at a single developmental stage. We found that genes pertaining to positive regulation of neural progenitor cell proliferation as well as known progenitor cell markers were upregulated in the midbrain prior to metamorphic climax; concurrently, expression of cell cycle timing regulators decreased across this period, supporting the notion that cell cycle lengthening contributes to a decrease in proliferation by the end of metamorphosis. We also found that at the start of metamorphosis, neural progenitor populations appeared to be similar across the fore-, mid-, and hindbrain regions. Genes pertaining to negative regulation of differentiation were upregulated in the spinal cord compared to the rest of the brain, however, suggesting that a different program may regulate neurogenesis there. Finally, we found that regulation of biological processes like cell fate commitment and synaptic signaling follow similar trajectories in the brain across early tadpole metamorphosis and mid- to late-embryonic mouse development. By comparing expression across both temporal and spatial conditions, we have been able to illuminate cell type and biological pathway dynamics in the brain during metamorphosis.
... It was well known that Wnt/β-catenin signaling pathway was essential for neural development [33]. Some studies showed that β-catenin, a key transcriptional regulator, was closely relate with NTDs occurrence [34]. ...
Background: S-adenosylmethionine as a major methyl donor play a key role in methylation modification in vivo, and its disorder was closely related to neural tube defects. However, the underlying mechanism between SAM deficiency and NTDs remained unclear.
Methods: we investigated the association between histone methylation modification and Wnt/β-catenin signaling pathway in NTDs induced by SAM deficiency. The levels of SAM and SAH were determined by enzyme linked immunosorbent assay. The expressions of H3K27me3 and Wnt/β-catenin signaling pathway specific markers were demonstrated by western blotting, reverse transcription, and quantitative PCR and immunofluorescence in ethionine induced E11.5 mouse NTDs and NSCs models.
Results: we found that the incidence rate of NTDs induced by ethionine were 46.2%, post treatment of ethionine combined with SAM, the incidence rate of NTDs was reduced to 26.2%. The level of SAM was significantly decreased (P<0.05) and a reduction in the SAM/SAH ratio was observed. The SAM depletion caused the reduction of both H3K27me3 modifications and UTX activity, and inhibited the marker proteins (β-catenin, TCF-4, Axin-2, p-GSK-3β, CyclinD1, and C-myc) in Wnt/β-catenin signaling pathway (P<0.05). The differentiations of neural stem cells into neurons and oligodendrocytes were inhibited under SAM deficiency (P<0.05).
Conclusions: These results indicated that the depletion of SAM led to reduced H3K27me3 modifications, prevented the activation of Wnt/β-catenin signaling pathway and NSCs differentiation, which provided an understanding of the novel function of epigenetic regulation in NTDs.
