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A Sonic hedgehog (Shh) response deficit in trisomic cells may be a common denominator for multiple features of Down syndrome
Abstract
The hedgehog (HH) family of growth factors is involved in many aspects of growth and development, from the establishment of left-right axes at gastrulation to the patterning and formation of multiple structures in essentially every tissue, to the maintenance and regulation of stem cell populations in adults. Sonic hedgehog (Shh) in particular acts as a mitogen, regulating proliferation of target cells, a growth factor that triggers differentiation in target populations, and a morphogen causing cells to respond differently based on their positions along a spatial and temporal concentration gradient. Given its very broad range of effects in development, it is not surprising that many of the structures affected by a disruption in Shh signaling are also affected in Down syndrome (DS). However, recent studies have shown that trisomic cerebellar granule cell precursors have a deficit, compared to their euploid counterparts, in their response to the mitogenic effects of Shh. This deficit substantially contributes to the hypocellular cerebellum in mouse models that parallels the human DS phenotype and can be corrected in early development by a single exposure to a small-molecule agonist of the Shh pathway. Here, we consider how an attenuated Shh response might affect several aspects of development to produce multiple phenotypic outcomes observed in DS.
... The significance of SHH in human brain development is illustrated by the dramatic consequences of SHH-pathway gene disruption, which include holoprosencephaly, seizure disorders, language or cognitive impairment, Down syndrome, hyperactivity, and schizophrenia (Heussler et al. 2002;Nanni et al. 1999;Belloni et al. 1996;Odent et al. 1999;Santiago et al. 2006;Currier et al. 2012;Betcheva et al. 2013). Many of these conditions are the result of SHH haploinsufficiency, thus highlighting the importance of SHH gene dosage in humans (Chiang et al. 1996). ...
... SHH is crucial for human brain development and changes in its signaling lead to distinct neuropathologies (Heussler et al. 2002;Nanni et al. 1999;Belloni et al. 1996;Odent et al. 1999;Santiago et al. 2006;Currier et al. 2012). Despite its importance, information on SHH expression profile in the developing human brain is still fragmentary. ...
Sonic Hedgehog (Shh) plays an instrumental role in brain development, fine-tuning processes such as cell proliferation, patterning, and fate specification. Although, mutations in the SHH pathway in humans are associated with various neurodevelopmental disorders, ranging from holoprosencephaly to schizophrenia, its expression pattern in the developing human brain is not well established. We now determined the previously not reported wide expression of SHH in the human fetal cerebral cortex during most of the gestation period (10–40 gestational weeks). This spatiotemporal distribution puts Shh in a position to influence the fundamental processes involved in corticogenesis. SHH expression increased during development, shifting from progenitor cells in the proliferative zones to neurons, both glutamatergic and GABAergic, and astrocytes in upper cortical compartments. Importantly, the expression of its downstream effectors and complementary receptors revealed evolutionary differences in SHH-pathway gene expression between humans and rodents.
Electronic supplementary material
The online version of this article (10.1007/s00429-018-1621-5) contains supplementary material, which is available to authorized users.
... Certain brain and craniofacial abnormalities found in individuals with Down syndrome (DS), a developmental disorder caused by trisomy of human chromosome 21 (Hsa21), have been attributed to disruptions in sonic hedgehog (SHH) signaling (Roper et al., 2006(Roper et al., , 2009Trazzi et al., 2011;Currier et al., 2012;Das et al., 2013;Dutka et al., 2015). Mouse models of DS, such as Ts65Dn, have been used to investigate the mechanisms underlying the disabilities associated with DS (Davisson et al., 1993;Das and Reeves, 2011). ...
... Trisomic granule cell neuron precursors and cells of the first pharyngeal arch have a reduced response to SHH compared with their euploid counterparts (Currier et al., 2012). Here, we tested whether that was the case for characteristic dysmorphologies of the trisomic facial skeleton. ...
Background:
In Ts65Dn, a mouse model of Down syndrome (DS), brain and craniofacial abnormalities that parallel those in people with DS are linked to an attenuated cellular response to sonic hedgehog (SHH) signaling. If a similarly reduced response to SHH occurs in all trisomic cells, then chronic up-regulation of the pathway might have a positive effect on development in trisomic mice, resulting in amelioration of the craniofacial anomalies.
Results:
We crossed Ts65Dn with Ptch1(tm1Mps/+) mice and quantified the craniofacial morphology of Ts65Dn;Ptch(+/-) offspring to assess whether a chronic up-regulation of the SHH pathway rescued DS-related anomalies. Ts65Dn;Ptch1(+/-) mice experience a chronic increase in SHH in SHH-receptive cells due to haploinsufficiency of the pathway suppressor, Ptch1. Chronic up-regulation had minimal effect on craniofacial shape and did not correct facial abnormalities in Ts65Dn;Ptch(+/-) mice. We further compared effects of this chronic up-regulation of SHH to acute pathway stimulation in mice treated on the day of birth with a SHH pathway agonist, SAG. We found that SHH affects facial morphology differently based on chronic vs. acute postnatal pathway up-regulation.
Conclusions:
Our findings have implications for understanding the function of SHH in craniofacial development and for the potential use of SHH-based agonists to treat DS-related abnormalities. This article is protected by copyright. All rights reserved.
... The Hh pathway activity is dampened in cells derived from Down syndrome patients 10 and in mouse models of this syndrome 11 . Importantly, certain morphological and cognitive deficits associated with Down spectrum could be ameliorated by the application of a synthetic Hh agonist 11,12 , suggesting a general Hh pathway suppression in these patients. This assumption is paradoxical as the DYRK1A kinase has been described as a stimulator of Hh pathway activity 13,14 , which is expected to lead to an increase in Hh signalling. ...
... In conclusion, the data presented provide a mechanistic framework on the potential molecular reasons behind the reduced Hh sensitivity in Down patients 10,12 and why some of the Down-related clinical syndromes could be ameliorated by addition of the SMO agonist SAG 11 . Furthermore, in light of our data showing that DYRK1A negatively regulates the oncogenic Hh pathway, it is well conceivable why this gene has been described as a tumour suppressor in, for example, medulloblastoma 20 . ...
The Down syndrome-associated DYRK1A kinase has been reported as a stimulator of the developmentally important Hedgehog (Hh) pathway, but cells from Down syndrome patients paradoxically display reduced Hh signalling activity. Here we find that DYRK1A stimulates GLI transcription factor activity through phosphorylation of general nuclear localization clusters. In contrast, in vivo and in vitro experiments reveal that DYRK1A kinase can also function as an inhibitor of endogenous Hh signalling by negatively regulating ABLIM proteins, the actin cytoskeleton and the transcriptional co-activator MKL1 (MAL). As a final effector of the DYRK1A-ABLIM-actin-MKL1 sequence, we identify the MKL1 interactor Jumonji domain demethylase 1A (JMJD1A) as a novel Hh pathway component stabilizing the GLI1 protein in a demethylase-independent manner. Furthermore, a Jumonji-specific small-molecule antagonist represents a novel and powerful inhibitor of Hh signal transduction by inducing GLI1 protein degradation in vitro and in vivo.
... This hypothesis is supported by the demonstration that a single acute SHH pathway stimulation can overcome a mitogenic deficit in cerebellar precursor neurons and normalize cerebellar structure in Ts6Dn mice (Vaillant and Monard, 2009). Ptch1 is the main repressor of the canonical SHH pathway (Currier et al., 2012). To test our hypothesis that a SHH response deficit underlies multiple DS phenotypes, we utilized a genetic model of widespread pathway up-regulation, the Ptch1 tm1Mps/+ (Ptch1 +/− ) mouse, which carries a LacZ "knockin" to the first and second exons of Ptch1 (Goodrich et al., 1997). ...
... Cumulatively, our results are not consistent with a deficit in response to SHH in all trisomic cell populations in the CNS (Currier et al., 2012) (Appendix: Supplementary Table S1). The behavior results suggest that only some cell types in the CNS evidence an amelioration of trisomic phenotypes whereas others were not affected or were further impaired. ...
Down Syndrome (DS) is a highly complex developmental genetic disorder caused by trisomy for human chromosome 21 (Hsa21). All individuals with DS exhibit some degree of brain structural changes and cognitive impairment; mouse models such as Ts65Dn have been instrumental in understanding the underlying mechanisms. Several phenotypes of DS might arise from a reduced response of trisomic cells to the Sonic Hedgehog (SHH) growth factor. If all trisomic cells show a similar reduced response to SHH, then up-regulation of the pathway in trisomic cells might ameliorate multiple DS phenotypes. We crossed Ptch1(tm1Mps)(/+) mice, in which the canonical SHH pathway is expected to be up-regulated in every SHH-responsive cell due to the loss of function of one allele of the pathway suppressor, Ptch1, to the Ts65Dn DS model and assessed the progeny for possible rescue of multiple DS-related phenotypes. Down-regulation of Ptch produced several previously unreported effects on development by itself, complicating interpretation of some phenotypes, and a number structural or behavioral effects of trisomy were not compensated by SHH signaling. However, a deficit in a nest-building task was partially restored in Ts;Ptch(+/-) mice, as were structural anomalies of the cerebellum in Ts65Dn mice. These results extend the body of evidence indicating that reduced response to SHH in trisomic cells and tissues contributes to various aspects of the trisomic phenotype.
Copyright © 2014. Published by Elsevier Ireland Ltd.
... Relevant to our studies, PRC2 deficiency is associated with impaired progression from OPCs to myelinating OLs [160]. Even though the dysregulation of the SHH pathway and enhanced expression of the PTCH1 receptor in DS [209,223,252,253] has been attributed, at least partially, to the triplication of APP [252], both SHH and PTCH1 are targets for transcriptional repression by PRC2 members [68,236], and dysregulation of the SHH pathway can be caused by increased transcription of key pathway genes due to the increased dosage of HMGN1 and alleviation of PRC2 repression. Similarly to neuronal dysregulation, the perturbation in glial cells and diminished maturation of oligodendrocytes can be potentially linked to a deficient silencing of the genes necessary for proper lineage development and functional maturity. ...
Intellectual disability is a well-known hallmark of Down Syndrome (DS) that results from the triplication of the critical region of human chromosome 21 (HSA21). Major studies were conducted in recent years to gain an understanding about the contribution of individual triplicated genes to DS-related brain pathology. Global transcriptomic alterations and widespread changes in the establishment of neural lineages, as well as their differentiation and functional maturity, suggest genome-wide chromatin organization alterations in trisomy. High Mobility Group Nucleosome Binding Domain 1 (HMGN1), expressed from HSA21, is a chromatin remodeling protein that facilitates chromatin decompaction and is associated with acetylated lysine 27 on histone H3 (H3K27ac), a mark correlated with active transcription. Recent studies causatively linked overexpression of HMGN1 in trisomy and the development of DS-associated B cell acute lymphoblastic leukemia (B-ALL). HMGN1 has been shown to antagonize the activity of the Polycomb Repressive Complex 2 (PRC2) and prevent the deposition of histone H3 lysine 27 trimethylation mark (H3K27me3), which is associated with transcriptional repression and gene silencing. However, the possible ramifications of the increased levels of HMGN1 through the derepression of PRC2 target genes on brain cell pathology have not gained attention. In this review, we discuss the functional significance of HMGN1 in brain development and summarize accumulating reports about the essential role of PRC2 in the development of the neural system. Mechanistic understanding of how overexpression of HMGN1 may contribute to aberrant brain cell phenotypes in DS, such as altered proliferation of neural progenitors, abnormal cortical architecture, diminished myelination, neurodegeneration, and Alzheimer’s disease-related pathology in trisomy 21, will facilitate the development of DS therapeutic approaches targeting chromatin.
... This impaired responsiveness to the SHH pathway has been reported in the Ts65Dn mouse model of DS (Roper et al., 2006;Trazzi et al., 2011;Currier et al., 2012), and we show that this deficit is present in human differentiated NPCs as well. Here we also report the first phenotypic correction in human cells by modulating the SHH pathway. ...
The intellectual disability found in people with Down syndrome is associated with numerous changes in early brain development, including the proliferation and differentiation of neural progenitor cells (NPCs) and the formation and maintenance of myelin in the brain. To study how early neural precursors are affected by trisomy 21, we differentiated two isogenic lines of induced pluripotent stem cells derived from people with Down syndrome into brain-like and spinal cord-like NPCs and promoted a transition towards oligodendroglial fate by activating the Sonic hedgehog (SHH) pathway. In the spinal cord-like trisomic cells, we found no difference in expression of OLIG2 or NKX2.2, two transcription factors essential for commitment to the oligodendrocyte lineage. However, in the brain-like trisomic NPCs, OLIG2 is significantly upregulated and is associated with reduced expression of NKX2.2. We found that this gene dysregulation and block in NPC transition can be normalized by increasing the concentration of a SHH pathway agonist (SAG) during differentiation. These results underscore the importance of regional and cell type differences in gene expression in Down syndrome and demonstrate that modulation of SHH signaling in trisomic cells can rescue an early perturbed step in neural lineage specification.
... This impaired responsiveness to the SHH pathway has been reported before in the Ts65Dn mouse model of DS (Currier et al., 2012;Roper et al., 2006;Trazzi et al., 2011), and we show that this deficit is present in human differentiated NPCs as well. However, previous work and our transcriptome profiling indicates that this SHH pathway dysregulation and dysfunction is not due to the triplication of SHH pathway genes on HSA21. ...
The intellectual disability found in people with Down syndrome (DS) is associated with a decrease in white matter in the central nervous system. To study the mechanism of this myelination deficit, we differentiated two isogenic lines of induced pluripotent stem cells (iPSCs) derived from people with DS into brain-like and spinal cord-like neural progenitor cells (NPCs) and promoted a transition towards oligodendroglial fate by activating the Sonic hedgehog (SHH) pathway. In the spinal cord-like trisomic cells, we found no difference in expression of OLIG2 or NKX2.2, two transcription factors essential for commitment to the oligodendrocyte (OL) lineage. However, in the brain-like trisomic NPCs, OLIG2 is significantly upregulated and is associated with reduced expression of NKX2.2. We found that this gene dysregulation and block in NPC transition can be normalized by increasing the concentration of a SHH pathway agonist (SAG) during differentiation. These results underscore the importance of regional and cell type differences in gene expression in DS and demonstrate that modulation of SHH signaling in trisomic cells can rescue an early perturbed step in neural lineage specification in DS.
... Notably, the reduction in neuronal differentiation is believed to be caused by a gliogenic shift, meaning that neuroprogenitor cells alter their differentiation ability in favor of astrocytes rather than neurons (Guidi et al., 2008;Zdaniuk et al., 2011;Chen et al., 2014). Several mechanisms can cause the gliogenic shift in DS, among which is a decrease in progenitor cell proliferation (Roper et al., 2006;Contestabile et al., 2007;Trazzi et al., 2013) and deficits in the Sonic hedgehog signaling pathway which have been directly shown to cause a reduction in the production of neurons (Roper et al., 2006;Currier et al., 2012;Das et al., 2013;Trazzi et al., 2013). Remarkably, studies have shown that correcting these deficits can rescue neuronal number in a DS animal model (Das et al., 2013). ...
Down Syndrome (DS) is the most common genetic cause of intellectual disability in which delays and impairments in brain development and function lead to neurological and cognitive phenotypes. Traditionally, a neurocentric approach, focusing on neurons and their connectivity, has been applied to understanding the mechanisms involved in DS brain pathophysiology with an emphasis on how triplication of chromosome 21 leads to alterations in neuronal survival and homeostasis, synaptogenesis, brain circuit development, and neurodegeneration. However, recent studies have drawn attention to the role of non-neuronal cells, especially astrocytes, in DS. Astrocytes comprise a large proportion of cells in the central nervous system (CNS) and are critical for brain development, homeostasis, and function. As triplication of chromosome 21 occurs in all cells in DS (with the exception of mosaic DS), a deeper understanding of the impact of trisomy 21 on astrocytes in DS pathophysiology is warranted and will likely be necessary for determining how specific brain alterations and neurological phenotypes emerge and progress in DS. Here, we review the current understanding of the role of astrocytes in DS, and discuss how specific perturbations in this cell type can impact the brain across the lifespan from early brain development to adult stages. Finally, we highlight how targeting, modifying, and/or correcting specific molecular pathways and properties of astrocytes in DS may provide an effective therapeutic direction given the important role of astrocytes in regulating brain development and function.
... Previous evidence demonstrated the mutations in genes of HH signaling were implicated in the occurrence of CHD with a phenotype of ASD, VSD or AVSD, and the responsible mutated genes include SHH, Gli3 and MKS1. [80][81][82][83] Noonan syndrome, one of the most common genetic syndromes of CHD, is caused by mutations in genes of the RAS-MAPK pathway. 84 At present, several genes have been verified to be responsible for the development of Noonan syndrome and other disorders. ...
Congenital heart disease (CHD) is the most common of congenital cardiovascular malformations associated with birth defects, and it results in significant morbidity and mortality worldwide. The classification of CHD is still elusive owing to the complex pathogenesis of CHD. Advances in molecular medicine have revealed the genetic basis of some heart anomalies. Genes associated with CHD might be modulated by various epigenetic factors. Thus, the genetic and epigenetic factors are gradually accepted as important triggers in the pathogenesis of CHD. However, few literatures have comprehensively elaborated the genetic and epigenetic mechanisms of CHD. This review focuses on the etiology of CHD from genetics and epigenetics to discuss the role of these factors in the development of CHD. The interactions between genetic and epigenetic in the pathogenesis of CHD are also elaborated. Chromosome abnormalities and gene mutations in genetics, and DNA methylations, histone modifications and on-coding RNAs in epigenetics are summarized in detail. We hope the summative knowledge of these etiologies may be useful for improved diagnosis and further elucidation of CHD so that morbidity and mortality of children with CHD can be reduced in the near future.
... Moreover, active Hh signaling is involved in regeneration after lung injury and in the maintenance of physiological pulmonary tissue quiescence [62]. In addition, Hh signaling is suppressed in Down Syndrome patients and pharmacological induction alleviates major symptoms in mouse models, suggesting novel areas of potential Hh agonist treatments [33,[63][64][65]. ...
Simple Summary
Elevated GLI1 expression levels are associated with improved survival in NB patients and GLI1 overexpression exerts tumor-suppressive traits in cultured NB cells. However, NB cells are protected from increased GLI1 levels as they have lost the ability to form primary cilia and transduce Hedgehog signals. This study identifies an isoxazole (ISX) molecule with primary cilia-independent GLI1-activating properties, which blocks NB cell growth. Mechanistically, ISX combines the removal of GLI3 repressor and the inhibition of class I HDACs, providing proof-of-principle evidence that small molecule-mediated activation of GLI1 could be harnessed therapeutically in the future.
Abstract
Although being rare in absolute numbers, neuroblastoma (NB) represents the most frequent solid tumor in infants and young children. Therapy options and prognosis are comparably good for NB patients except for the high risk stage 4 class. Particularly in adolescent patients with certain genetic alterations, 5-year survival rates can drop below 30%, necessitating the development of novel therapy approaches. The developmentally important Hedgehog (Hh) pathway is involved in neural crest differentiation, the cell type being causal in the etiology of NB. However, and in contrast to its function in some other cancer types, Hedgehog signaling and its transcription factor GLI1 exert tumor-suppressive functions in NB, rendering GLI1 an interesting new candidate for anti-NB therapy. Unfortunately, the therapeutic concept of pharmacological Hh/GLI1 pathway activation is difficult to implement as NB cells have lost primary cilia, essential organelles for Hh perception and activation. In order to bypass this bottleneck, we have identified a GLI1-activating small molecule which stimulates endogenous GLI1 production without the need for upstream Hh pathway elements such as Smoothened or primary cilia. This isoxazole compound potently abrogates NB cell proliferation and might serve as a starting point for the development of a novel class of NB-suppressive molecules.
... In previous studies, HSCR has been shown to involve Down's syndrome (HSCR-DS) (14). DS is known to involve cerebellar hypoplasia in addition to impaired development of the ENS and hearing loss (15). In a previous study, a significant association of endothelin receptor B (EDNRB) with HSCR-DS was shown in humans (16). ...
The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a Smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS.
... Le syndrome de Down, l'une des causes génétiques les plus fréquentes de déficit intellectuel, est relié dans la majorité des cas à une insuffisance d'activité de la signalisation Shh par surexpression du récepteur Ptc vraisemblablement liée à des niveaux accrus du précurseur de la protéine amyloïde (Trazzi et al., 2013). L'activation pharmacologique de la voie par un agoniste de Smo suffit à normaliser la morphologie du cervelet et à restaurer la capacité d'apprentissage associée au fonctionnement de l'hippocampe (Currier et al., 2012;Das et al., 2013). ...
Au cours des dernières années, des progrès significatifs ont été réalisés dans la prévention des épisodes de démyélinisation qui surviennent au cours de la sclérose en plaques, mais beaucoup moins dans le domaine de la régénération de la myéline détruite. Notre équipe a précédemment démontré que la protéine Sonic Hedgehog (Shh) est un régulateur positif des progéniteurs oligodendrocytaires favorisant la réparation de la myéline dans un modèle de démyélinisation induite par injection focale de lysolécithine dans le corps calleux chez la souris. Cependant, les mécanismes moléculaires mis en jeu restent encore largement méconnus. Mon projet de thèse consiste à explorer le rôle d'un co-récepteur de Shh, Boc, une glycoprotéine reliée aux molécules d'adhésion cellulaire (CAM) et fortement induite à l’issue d’une démyélinisation. Dans un premier temps, nous avons focalisé nos investigations sur la caractérisation du mutant Boc knockout au cours de la myélinisation développementale. Nos résultats révèlent des activités région et temps spécifiques de Boc sur les mécanismes de prolifération et de maturation des progéniteurs oligodendrocytaires. De plus, les souris Boc knockout présentent un phénotype d’hypermyélinisation qui apparaît au cours des premières semaines post-natales et persiste à l’âge adulte.Dans un second temps, nous avons déterminé l'effet de l'absence de Boc sur les processus de démyélinisation et de réparation de la myéline. En utilisant le modèle d’injection focale de lysolécithine dans le corps calleux, nous avons démontré un retard dans la maturation des progéniteurs oligodendrocytaires et une réduction de la régression des lésions chez les souris dépourvues de Boc.Nos résultats indiquent ainsi un rôle positif de Boc dans le processus de régénération faisant suite à une démyélinisation. Par ailleurs, l’absence de Boc diminue la réaction gliale inflammatoire étroitement associée au mécanisme de régénération de la myéline. L’ensemble de nos résultats indique une activité différente du récepteur au cours des processus de myélinisation et de remyélinisation du système nerveux central. Ces données originales contribuent à la caractérisation d’une composante de la signalisation Hedgehog impliquée dans le processus régénératif associé aux pathologies démyélinisantes. Ces résultats seront importants à considérer dans le cadre de l’approche thérapeutique des maladies démyélinisantes.
... Granular cell precursors in the cerebellum as well as neural crest cells of Ts65Dn mice were found to have reduced response to the mitogenic factor Shh leading to cerebellar hypo-cellularity, craniofacial dysmorphism and a hypoplastic dorsal mesenchymal protrusion that leads to an atrioventricular septal defect Currier et al., 2012). The mTOR pathway implicated in cell growth and survival was found to be deregulated in the hippocampus of Ts1Cje and Dp(10)1Yey mice, in the brain of Tc1 mice and in the frontal cortex from DS human autopsies (Ahmed et al., 2013;Block et al., 2015;Perluigi et al., 2014;Troca-Marín et al., 2014) and has been associated with the AD pathology (Di Domenico et al., 2018). ...
The genotype-phenotype relationship and the physiopathology of Down Syndrome (DS) have been explored in the last 20 years with more and more relevant mouse models. From the early age of transgenesis to the new CRISPR/CAS9-derived chromosomal engineering and the transchromosomic technologies, mouse models have been key to identify homologous genes or entire regions homologous to the human chromosome 21 that are necessary or sufficient to induce DS features, to investigate the complexity of the genetic interactions that are involved in DS and to explore therapeutic strategies. In this review we report the new developments made, how genomic data and new genetic tools have deeply changed our way of making models, extended our panel of animal models, and increased our understanding of the neurobiology of the disease. But even if we have made an incredible progress which promises to make DS a curable condition, we are facing new research challenges to nurture our knowledge of DS pathophysiology as a neurodevelopmental disorder with many comorbidities during ageing.
... Meta-analysis of human and/or murine data revealed perturbation of some pathways involved in many cellular and molecular processes such as cell cycle, cytoskeleton organization, apoptosis, energy metabolism, cellular stress response, immune response, proteasomal activity, transcriptional activity, DNA repair or epigenetic mechanisms (Vilardell et al., 2011, Pelleri et al., 2018. Granular cell precursors in the cerebellum as well as neural crest cells of Ts65Dn mice were found to have reduced response to the mitogenic factor Shh leading to cerebellar hypocellularity, craniofacial dysmorphism and a hypoplastic dorsal mesenchymal protrusion that leads to an atrioventricular septal defect , Currier et al., 2012. The mTOR pathway implicated in cell growth and survival was found to be deregulated in the hippocampus of Ts1Cje and Dp(10)1Yey mice, in the brain of Tc1 mice and in the frontal cortex from DS human autopsies (Troca-Marín et al., 2014, Block et al., 2015, Ahmed et al., 2013, Perluigi et al., 2014 and has been associated with the AD pathology (Di Domenico et al., 2018). ...
... In individuals with Down syndrome (Trisomy 21), a characteristic craniofacial phenotype develops from an early developmental stage. Recent studies from the laboratory of Roger Reeves have linked this phenotype to reduced delamination, migration, and subsequent proliferation of neural crest cells, which appears to be due to reduced responsiveness of these cells to Shh [104,105]. Holoprosencephaly and fetal alcohol syndrome are discussed in Sections 5 and 9 of this review in greater detail. ...
Since its initial discovery in a Drosophila mutagenesis screen, the Hedgehog pathway has been revealed to be instrumental in the proper development of the vertebrate face. Vertebrates possess three hedgehog paralogs: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Of the three, Shh has the broadest range of functions both in the face and elsewhere in the embryo, while Ihh and Dhh play more limited roles. The Hedgehog pathway is instrumental from the period of prechordal plate formation early in the embryo, until the fusion of the lip and secondary palate, which complete the major patterning events of the face. Disruption of Hedgehog signaling results in an array of developmental disorders in the face, ranging from minor alterations in the distance between the eyes to more serious conditions such as severe clefting of the lip and palate. Despite its critical role, Hedgehog signaling seems to be disrupted through a number of mechanisms that may either be direct, as in mutation of a downstream target of the Hedgehog ligand, or indirect, such as mutation in a ciliary protein that is otherwise seemingly unrelated to the Hedgehog pathway. A number of teratogens such as alcohol, statins and steroidal alkaloids also disrupt key aspects of Hedgehog signal transduction, leading to developmental defects that are similar, if not identical, to those of Hedgehog pathway mutations. The aim of this review is to highlight the variety of roles that Hedgehog signaling plays in developmental disorders of the vertebrate face.
... Many types of refined screens with greater sensitivity and specificity are possible, taking advantage of transgenically marked zebrafish lines to ask specific questions about development of specific structures. Furthermore, our identification of phenotypes associated with pathways previously identified as central to multiple manifestations of trisomy, such as Shh signaling (Roper et al. 2009;Currier et al. 2012;Das et al. 2013), support the use of D. rerio for this type of large-scale systematic screen. This system represents a useful screening tool to identify individual candidate genes that may be significant drivers of DS phenotypes. ...
Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked loss-of-function of C. elegans orthologues of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for dosage sensitive effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS.
... As mentioned before, a study showed a relation between a decreased expression of the HH target gene GLI1 and the appearance of VSDs in Down syndrome patients [91], indicating that reduced HH signalling provokes VSDs in these patients. This hypothesis is supported by the fact that there are significant similarities between the heart phenotypes that are observed in HH signalling mutant mice and those seen in Down syndrome mouse models [147]. In regard to the development of VSDs, it is remarkable that a hypoplastic dorsal mesenchymal protrusion (a tissue derived from the second heart field) was found in Down syndrome patients and mouse models [148][149][150]. ...
An incomplete septation of the ventricles in the vertebrate heart that disturbes the strict separation between the contents of the two ventricles is termed a ventricular septal defect (VSD). Together with bicuspid aortic valves, it is the most frequent congenital heart disease in humans. Until now, life-threatening VSDs are usually treated surgically. To avoid surgery and to develop an alternative therapy (e.g., a small molecule therapy), it is necessary to understand the molecular mechanisms underlying ventricular septum (VS) development. Consequently, various studies focus on the investigation of signalling pathways, which play essential roles in the formation of the VS. In the past decade, several reports found evidence for an involvement of Hedgehog (HH) signalling in VS development. In this review article, we will summarise the current knowledge about the association between HH signalling and VS formation and discuss the use of such knowledge to design treatment strategies against the development of VSDs.
... In vivo treatment with EGCG at E7 and E8, around the time of the developing NCC deficit, appears to improve some of the NCC embryonic dysmorphology, especially in PA1 volume and NCC number in Ts65Dn E9.5 embryos. Gene expression was also altered by EGCG treatment and essentially normalized Rcan1, Shh, Ptch1 and Ets2 RNA expression-all of which are genes hypothesized to be related to some DS craniofacial deficits (18,38,39,48). EGCG treatment and Dyrk1a activity have been shown to be connected to and modulate the Shh pathway (49)(50)(51). ...
Trisomy 21 (Ts21) affects craniofacial precursors in individuals with Down syndrome (DS). The resultant craniofacial features in all individuals with Ts21 may significantly affect breathing, eating and speaking. Using mouse models of DS, we have traced the origin of DS-associated craniofacial abnormalities to deficiencies in neural crest cell (NCC) craniofacial precursors early in development. Hypothetically, three copies of Dyrk1a, a trisomic gene found in most humans with DS and mouse models of DS, may significantly affect craniofacial structure. We hypothesized that we could improve DS-related craniofacial abnormalities in mouse models using a Dyrk1a inhibitor or by normalizing Dyrk1a gene dosage. In vitro and in vivo treatment with Epigallocatechin gallate (EGCG), a Dyrk1a inhibitor, modulated trisomic NCC deficiencies at embryonic time points. Furthermore, prenatal EGCG treatment normalized some craniofacial phenotypes, including cranial vault in adult Ts65Dn mice. Normalization of Dyrk1a copy number in an otherwise trisomic Ts65Dn mice normalized many dimensions of the cranial vault, but did not correct all craniofacial anatomy. These data underscore the complexity of the gene-phenotype relationship in trisomy and suggest that changes in Dyrk1a expression play an important role in morphogenesis and growth of the cranial vault. These results suggest that a temporally specific prenatal therapy may be an effective way to ameliorate some craniofacial anatomical changes associated with DS.
... The left-right signaling pathway is affected when trisomy and Tbx5 haploinsufficiency are combined. We suggested previously that a universal response deficit to Sonic hedgehog signaling due to trisomy could explain many of the clinical features of DS [44]; hedgehog signaling plays a regulatory role in left-right patterning and can regulate Pitx2 expression [45][46][47]. Expression of Sonic hedgehog in the second heart field is essential for proper growth of the dorsal mesenchymal protrusion, without which AVSD occurs [48]. ...
Background:
Nearly half of all individuals with Down Syndrome (DS) have some type of congenital heart defect (CHD), suggesting that DS sensitizes to CHD but does not cause it. We used a common mouse model of DS, the Ts65Dn mouse, to study the contribution of Tbx5, a known modifier of CHD, to heart defects on a trisomic backgroun. Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10% formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays.
Methods:
Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10% formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays.
Results:
We crossed mice that were heterozygous for a Tbx5 null allele with Ts65Dn mice. Mice that were trisomic and carried the Tbx5 mutation (Ts65Dn;Tbx5 (+/-) ) had a significantly increased incidence of overriding aorta compared to their euploid littermates. Ts65Dn;Tbx5 (+/-) mice also showed reduced expression of Pitx2, a molecular marker for the left atrium. Transcript levels of the trisomic Adamts1 gene were decreased in Tbx5 (+/-) mice compared to their euploid littermates. Evidence of a valid binding site for TBX5 upstream of the trisomic Adamts1 locus was also shown.
Conclusion:
Haploinsufficiency of Tbx5 and trisomy affects alignment of the aorta and this effect may stem from deviations from normal left-right patterning in the heart. We have unveiled a previously unknown interaction between the Tbx5 gene and trisomy, suggesting a connection between Tbx5 and trisomic genes important during heart development.
... Ts65Dn is the most widely studied mouse model for DS (Das and Reeves, 2011). Cerebellar hypoplasia in Ts65Dn mice results from the reduced response of cerebellar granule cell precursors (GCPs) to the mitogenic effects of SHH, resulting in decreased numbers of granule cell (GC) and Purkinje cell neurons in the adult cerebellum (Dahmane and Ruiz-i-Altaba, 1999;Roper et al., 2006;Currier et al., 2012). Temporary upregulation of the HH pathway by a single injection of 20 μg/g SAG (μg SAG/g body weight) on the day of birth (P0) normalized cerebellar morphology in trisomic mice by postnatal day 6 (P6) (Roper et al., 2006). ...
Hedgehog (HH) signaling, particularly by sonic hedgehog (SHH), is implicated in several essential activities during morphogenesis, and its misexpression causes a number of developmental disorders in humans. In particular, a reduced mitogenic response of cerebellar granule cell precursors to SHH signaling in a mouse model for Down syndrome (DS), Ts65Dn, is substantially responsible for reduced cerebellar size. A single treatment of newborn trisomic mice with an agonist of the SHH pathway (SAG) normalized cerebellar morphology and restored some cognitive deficits, suggesting a possible therapeutic application of SAG for treating the cognitive impairments of DS. While the beneficial effects on the cerebellum are compelling, inappropriate activation of the HH pathway causes anomalies elsewhere in the head, particularly in the formation and patterning of the craniofacial skeleton. To determine whether an acute treatment of SAG has an effect on craniofacial morphology, we quantitatively analyzed the cranial form of adult euploid and Ts65Dn mice that were injected with either SAG or vehicle at birth. We found significant deformation of adult craniofacial shape in some animals that had received SAG at birth. The most pronounced differences between the treated and untreated mice were in the midline structures of the facial skeleton. The SAG-driven craniofacial dysmorphogenesis was dose-dependent and possibly incompletely penetrant at lower concentrations. Our findings illustrate that activation of HH signaling, even with an acute postnatal stimulation can lead to localized dysmorphology of the skull by generating modular shape changes in the facial skeleton. These observations have important implications for translating HH agonist-based treatments for DS.
Skeletal Class III (SCIII) is among the most challenging craniofacial dysmorphologies to treat. There is, however, a knowledge gap regarding which syndromes share this clinical phenotype. The aims of this study were to: (i) identify the syndromes affected by the SCIII phenotype; (ii) clarify the involvement of maxillary and/or mandibular structures; (iii) explore shared genetic/molecular mechanisms. A two-step strategy was designed: [Step#1] OMIM, MHDD, HPO, GeneReviews and MedGen databases were explored; [Step#2]: Syndromic conditions indexed in [Step#1] were explored in Medline, Pubmed, Scopus, Cochrane Library, WOS and OpenGrey. Eligibility criteria were defined. Individual studies were assessed for risk of bias using the New Ottawa Scale. For quantitative analysis, a meta-analysis was conducted. This scoping review is a hypothesis-generating research. Twenty-two studies met the eligibility criteria. Eight syndromes affected by the SCIII were targeted: Apert syndrome, Crouzon syndrome, achondroplasia, X-linked hypohidrotic ectodermal dysplasia (XLED), tricho-dento-osseous syndrome, cleidocranial dysplasia, Klinefelter and Down syndromes. Despite heterogeneity between studies [ p < 0.05], overall effects showed that midface components were affected in Apert and Down Syndromes, lower face in Klinefelter Syndrome and midface and lower face components in XLED. Our review provides new evidence on the craniofacial characteristics of genetically confirmed syndromes exhibiting the SCIII phenotype. Four major regulatory pathways might have a modulatory effect on this phenotype.
Impact
What does this review add to the existing literature? To date, there is no literature exploring which particular syndromes exhibit mandibular prognathism as a common trait.
Through this research, it was possibly to identify the particular syndromes that share the skeletal Class III phenotype (mandibular prognathism) as a common trait highlighting the common genetic and molecular pathways between different syndromes acknowledging their impact in craniofacial development.
Trisomy 21 and mutations in the Sonic hedgehog (SHH) signaling pathway cause overlapping and pleiotropic phenotypes including cerebellar hypoplasia, craniofacial abnormalities, congenital heart defects, and Hirschsprung disease. Trisomic cells derived from individuals with Down syndrome possess deficits in SHH signaling, suggesting that overexpression of chromosome 21 genes may contribute to SHH-associated phenotypes by disrupting normal SHH signaling during development. However, chromosome 21 does not encode any known components of the canonical SHH pathway. Here, we sought to identify chromosome 21 genes that modulate SHH signaling by overexpressing 163 chromosome 21 cDNAs in a series of SHH-responsive cell lines. We confirmed overexpression of trisomic candidate genes using RNA-seq in Ts65Dn and TcMAC21 cerebellum. Our findings indicate that some chromosome 21 genes, including DYRK1A, upregulate SHH signaling whereas others, such as HMGN1, inhibit SHH signaling. Individual overexpression of four genes (B3GALT5, ETS2, HMGN1, and MIS18A) inhibits the SHH-dependent proliferation of primary granule cell precursors. Our study prioritizes dosage sensitive chromosome 21 genes for future mechanistic studies. Identifying which genes modulate SHH signaling may suggest new therapeutic avenues for ameliorating Down syndrome phenotypes.
Dysregulation of Sonic hedgehog (SHH) signaling may contribute to multiple Down syndrome-associated phenotypes, including cerebellar hypoplasia, congenital heart defects, craniofacial and skeletal dysmorphologies, and Hirschsprung disease. Granule cell precursors isolated from the developing cerebellum of Ts65Dn mice are less responsive to the mitogenic effects of SHH than euploid cells, and a single postnatal dose of the SHH pathway agonist SAG rescues cerebellar morphology and performance on learning and memory tasks in Ts65Dn mice. SAG treatment also normalizes expression levels of OLIG2 in neural progenitor cells derived from human trisomy 21 iPSCs. However, despite evidence that activating SHH signaling can ameliorate some Down syndrome-associated phenotypes, chromosome 21 does not encode any components of the canonical SHH pathway. Here, we screened 163 chromosome 21 cDNAs in a series of SHH-responsive cell lines to identify chromosome 21 genes that modulate SHH signaling. We confirmed overexpression of trisomic candidate genes using RNA-seq in Ts65Dn and TcMAC21 cerebellum. Our study indicates that some chromosome 21 genes, including DYRK1A , upregulate SHH signaling while others, such as HMGN1 and MIS18A , inhibit SHH signaling. Overexpression of genes involved in chromatin structure and mitosis, but not genes previously implicated in ciliogenesis, regulate the SHH pathway. Our data suggest that cerebellar hypoplasia and other phenotypes related to aberrant SHH signaling arise from the net effect of trisomy for multiple chromosome 21 genes rather than the overexpression of a single trisomic gene. Identifying which chromosome 21 genes modulate SHH signaling may suggest new therapeutic avenues for ameliorating Down syndrome phenotypes.
One Sentence Summary
Multiple chromosome 21 genes modulate Sonic hedgehog signaling, which is dysregulated in Down syndrome.
Trisomy 21, the source of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. We investigate how PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT accumulates around the centrosome as a cluster of enlarged cytoplasmic puncta that localize along microtubules (MTs) and at MT ends. Cytoplasmic PCNT puncta impact the density, stability and localization of the MT trafficking network required for primary cilia. The PCNT puncta appear to sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins, PCM1, CEP131 and CEP290, important for ciliogenesis, accumulate at enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta and pericentrosomal crowding, reestablish a normal density of MTs around the centrosome, and restore ciliogenesis to wild type levels. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the MT network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia.
Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked loss-of-function of C. elegans orthologues of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for dosage sensitive effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS.
Sonic hedgehog (Shh) signaling plays critical roles during early central nervous system development, such as neural cell proliferation, patterning of the neural tube and neuronal differentiation. While Shh signaling is still present in the postnatal brain, the roles it may play are, however, largely unknown. In particular, Shh signaling components are found at the synaptic junction in the maturing hippocampus during the first two postnatal weeks. This period is characterized by the presence of ongoing spontaneous synaptic activity at the cellular and network levels thought to play important roles in the onset of neuronal circuit formation and synaptic plasticity. Here, we demonstrate that non-canonical Shh signaling increases the frequency of the synchronized electrical activity called Giant Depolarizing Potentials (GDP) and enhances spontaneous GABA post-synaptic currents in the rodent hippocampus during the early postnatal period. This effect is mediated specifically through the Shh co-receptor Smoothened via intracellular Ca 2+ signal and the activation of the BDNF-TrkB signaling pathway. Given the importance of these spontaneous events on neuronal network maturation and refinement, this study opens new perspectives for Shh signaling on the control of early stages of postnatal brain maturation and physiology.
Children with trisomy 21 (Down syndrome [DS]) have a 130-fold increased incidence of Hirschsprung Disease (HSCR), a developmental defect where the enteric nervous system (ENS) is missing from distal bowel (i.e., distal bowel is aganglionic). Treatment for HSCR is surgical resection of aganglionic bowel, but many children have bowel problems after surgery. Post-surgical problems like enterocolitis and soiling are especially common in children with DS. To determine how trisomy 21 affects ENS development, we evaluated the ENS in two DS mouse models, Ts65Dn and Tc1. These mice are trisomic for many chromosome 21 homologous genes, including Dscam and Dyrk1a, which are hypothesized to contribute to HSCR risk. Ts65Dn and Tc1 mice have normal ENS precursor migration at E12.5 and almost normal myenteric plexus structure as adults. However, Ts65Dn and Tc1 mice have markedly reduced submucosal plexus neuron density throughout the bowel. Surprisingly, the submucosal neuron defect in Ts65Dn mice is not due to excess Dscam or Dyrk1a, since normalizing copy number for these genes does not rescue the defect. These findings suggest the possibility that the high frequency of bowel problems in children with DS and HSCR may occur because of additional unrecognized problems with ENS structure.
Trisomy 21 (T21) is the most prevalent human chromosomal disorder, causing a range of cardiovascular, musculoskeletal, and neurological abnormalities. However, the cellular processes disrupted by T21 are poorly understood. Consistent with the clinical overlap between T21 and ciliopathies, we discovered that T21 disrupts cilia formation and signaling. Cilia defects arise from increased expression of Pericentrin, a centrosome scaffold and trafficking protein encoded on chromosome 21. Elevated Pericentrin is necessary and sufficient for T21 cilia defects. Pericentrin accumulates at centrosomes and dramatically in the cytoplasm surrounding centrosomes. Centrosome Pericentrin recruits more γ-tubulin and enhances microtubules, whereas cytoplasmic Pericentrin assembles into large foci that do not efficiently traffic. Moreover, the Pericentrin-associated cilia assembly factor IFT20 and the ciliary signaling molecule Smoothened do not efficiently traffic to centrosomes and cilia. Thus, increased centrosome protein dosage produces ciliopathy-like outcomes in T21 cells by decreasing trafficking between the cytoplasm, centrosomes, and cilia.
Congenital heart disease is present in 44-56% of fetuses with Down syndrome (DS). There are, however, signs that hearts in DS without apparent structural heart defects also differ from those in the normal population. We aimed to compare the atrioventricular (AV) septum and valves in 3 groups: DS without AV septal defect (DS no-AVSD), DS with AVSD (DS AVSD) and control hearts.
The ventricular septum, membranous septum and AV valves were examined and measured in histological sections of 15 DS no-AVSD, 8 DS AVSD and 34 control hearts. In addition, the ventricular septum length was measured on ultrasound images of fetal (6 DS AVSD, 9 controls) and infant (10 DS no-AVSD, 10 DS AVSD, 10 controls) hearts.
The membranous septum was 3 times larger in DS no-AVSD fetuses compared to control fetuses, and valve dysplasia was frequently (64%) observed. The ventricular septum was shorter in patients with DS both with and without AVSD, as compared to the control group.
DS no-AVSD hearts are not normal as they have a larger membranous septum, shorter ventricular septum and dysplasia of the AV valves as compared to control hearts. © 2015 S. Karger AG, Basel.
The primary unseptated heart tube undergoes extensive remodeling including septation at the atrial, atrioventricular, ventricular, and ventriculo-arterial level. Alignment and fusion of the septal components is required to ensure full septation of the heart. Deficiencies lead to septal defects at various levels. Addition of myocardium and mesenchymal tissues from the second heart field (SHF) to the primary heart tube, as well as a population of neural crest cells, provides the necessary cellular players. Surprisingly, the study of the molecular background of these defects does not show a great diversity of responsible transcription factors and downstream gene pathways. Epigenetic modulation and mutations high up in several transcription factor pathways (e.g. NODAL and GATA4) may lead to defects at all levels. Disturbance of modulating pathways, involving primarily the SHF-derived cell populations and the genes expressed therein, results at the arterial pole (e.g. TBX1) in a spectrum of ventricular septal defects located at the level of the outflow tract. At the venous pole (e.g. TBX5), it can explain a variety of atrial septal defects. The various defects can occur as isolated anomalies or within families. In this review developmental, morphological, genetic, as well as epigenetic aspects of septal defects are discussed.
Since the discovery of the segment polarity gene Hedgehog in Drosophila three decades ago, our knowledge of Hedgehog signaling pathway has considerably improved and paved the way to a wide field of investigations in the developing and adult central nervous system. Its peculiar transduction mechanism together with its implication in tissue patterning, neural stem cell biology, and neural tissue homeostasis make Hedgehog pathway of interest in a high number of normal or pathological contexts. Consistent with its role during brain development, misregulation of Hedgehog signaling is associated with congenital diseases and tumorigenic processes while its recruitment in damaged neural tissue may be part of the repairing process. This review focuses on the most recent data regarding the Hedgehog pathway in the developing and adult central nervous system and also its relevance as a therapeutic target in brain and spinal cord diseases.
Humans with Down syndrome (DS) and Ts65Dn mice both show a reduced volume of the cerebellum due to a significant reduction in the density of granule neurons. Recently, cerebellar hypoplasia in Ts65Dn mice was rescued by a single treatment with SAG, an agonist of the Sonic hedgehog pathway, administered on the day of birth. In addition to normalizing cerebellar morphology, this treatment restored the ability to learn a spatial navigation task, which is associated with hippocampal function. It is not clear to what extent this improved performance results from restoration of the cerebellar architecture or a yet undefined role of Sonic hedgehog (Shh) in perinatal hippocampal development. The absence of a clearly demonstrated deficit in cerebellar function in trisomic mice exacerbates the problem of discerning how SAG acts to improve learning and memory. Here we show that phase reversal adaptation and consolidation of the vestibulo-ocular reflex is significantly impaired in Ts65Dn mice, providing for the first time a precise characterization of cerebellar functional deficits in this murine model of DS. However, these deficits do not benefit from the normalization of cerebellar morphology following treatment with SAG. Together with the previous observation that the synaptic properties of Purkinje cells are also unchanged by SAG treatment, this lack of improvement in a region-specific behavioral assay supports the possibility that a direct effect of Shh pathway stimulation on the hippocampus might explain the benefits of this potential approach to the improvement of cognition in DS.
Down syndrome (DS) is among the most frequent genetic causes of intellectual disability, and ameliorating this deficit is a major goal in support of people with trisomy 21. The Ts65Dn mouse recapitulates some major brain structural and behavioral phenotypes of DS, including reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus. We show that a single treatment of newborn mice with the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults. Further, SAG treatment at birth rescued phenotypes associated with hippocampal deficits that occur in untreated adult Ts65Dn mice. This treatment resulted in behavioral improvements and normalized performance in the Morris water maze task for learning and memory. SAG treatment also produced physiological effects and partially rescued both N-methyl-d-aspartate (NMDA) receptor-dependent synaptic plasticity and NMDA/AMPA receptor ratio, physiological measures associated with memory. These outcomes confirm an important role for the hedgehog pathway in cerebellar development and raise the possibility for its direct influence in hippocampal function. The positive results from this approach suggest a possible direction for therapeutic intervention to improve cognitive function for this population.
Mouse genetic models can be used to dissect molecular mechanisms that result in human disease. This approach requires detection and demonstration of compelling parallels between phenotypes in mouse and human. Ts65Dn mice are at dosage imbalance for many of the same genes duplicated in trisomy 21 or Down syndrome (DS), the most common live-born human aneuploidy. Analysis of the craniofacial skeleton of Ts65Dn mice using three-dimensional morphometric methods demonstrates an absolute correspondence between Ts65Dn and DS craniofacial dysmorphology, a distinctive and completely penetrant DS phenotype. The genes at dosage imbalance in Ts65Dn are localized to a small region of mouse chromosome 16 and, by comparative mapping, to the corresponding region of human Chromosome 21, providing independent experimental data supporting the contribution of genes in this region to this characteristic DS phenotype. This analysis establishes precise parallels in human and mouse skull phenotypes resulting from dosage imbalance for the same genes, revealing strong conservation of the evolved developmental genetic program that underlies mammalian skull morphology and validating the use of this mouse model in the analysis of this important DS phenotype. This evolutionary conservation further establishes the mouse as a valid model for a wide range of syndromes producing craniofacial maldevelopment. Dev Dyn;217:137–145. © 2000 Wiley-Liss, Inc.
Remarkable advances have been made in recent years towards therapeutics for cognitive impairment in individuals with Down syndrome (DS) by using mouse models. In this review, we briefly describe the phenotypes of mouse models that represent outcome targets for drug testing, the behavioral tests used to assess impairments in cognition and the known mechanisms of action of several drugs that are being used in preclinical studies or are likely to be tested in clinical trials. Overlaps in the distribution of targets and in the pathways that are affected by these diverse drugs in the trisomic brain suggest new avenues for DS research and drug development.
Down syndrome (DS) is the most common genetic cause of mental retardation and affects many aspects of brain development. DS individuals exhibit an overall reduction in brain size with a disproportionately greater reduction in cerebellar volume. The Ts65Dn mouse is segmentally trisomic for the distal 12-15 Mb of mouse chromosome 16, a region that shows perfect conserved linkage with human chromosome 21, and therefore provides a genetic model for DS. In this study, high resolution magnetic resonance imaging and histological analysis demonstrate precise neuro- anatomical parallels between the DS and the Ts65Dn cerebellum. Cerebellar volume is significantly reduced in Ts65Dn mice due to reduction of both the internal granule layer and the molecular layer of the cerebellum. Granule cell number is further reduced by a decrease in cell density in the internal granule layer. Despite these changes in Ts65Dn cerebellar structure, motor deficits have not been detected in several tests. Reduction in granule cell density in Ts65Dn mice correctly predicts an analogous pathology in humans; a significant reduction in granule cell density in the DS cerebellum is reported here for the first time. The candidate region of genes on chromosome 21 affecting cerebellar development in DS is therefore delimited to the subset of genes whose orthologs are at dosage imbalance in Ts65Dn mice, providing the first localization of genes affecting a neuroanatomical phenotype in DS. The application of this model for analysis of developmental perturbations is extended by the accurate prediction of DS cerebellar phenotypes.
An imbalance between inhibitory and excitatory neurotransmission has been proposed to contribute to altered brain function in individuals with Down syndrome (DS). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and accordingly treatment with GABA-A antagonists can efficiently restore cognitive functions of Ts65Dn mice, a genetic model for DS. However, GABA-A antagonists are also convulsant which preclude their use for therapeutic intervention in DS individuals. Here, we have evaluated safer strategies to release GABAergic inhibition using a GABA-A-benzodiazepine receptor inverse agonist selective for the α5-subtype (α5IA). We demonstrate that α5IA restores learning and memory functions of Ts65Dn mice in the novel-object recognition and in the Morris water maze tasks. Furthermore, we show that following behavioural stimulation, α5IA enhances learning-evoked immediate early gene products in specific brain regions involved in cognition. Importantly, acute and chronic treatments with α5IA do not induce any convulsant or anxiogenic effects that are associated with GABA-A antagonists or non-selective inverse agonists of the GABA-A-benzodiazepine receptors. Finally, chronic treatment with α5IA did not induce histological alterations in the brain, liver and kidney of mice. Our results suggest that non-convulsant α5-selective GABA-A inverse agonists could improve learning and memory deficits in DS individuals.
Human trisomy 21, the chromosomal basis of Down syndrome (DS), is the most common genetic cause of heart defects. Regions on human chromosome 21 (Hsa21) are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this study, we have analyzed the impact of duplications of each syntenic region on cardiovascular development in mice and have found that only the duplication on Mmu16, i.e., Dp(16)1Yey, is associated with heart defects. Furthermore, we generated two novel mouse models carrying a 5.43-Mb duplication and a reciprocal deletion between Tiam1 and Kcnj6 using chromosome engineering, Dp(16Tiam1-Kcnj6)Yey/+ and Df(16Tiam1-Kcnj6)Yey/+, respectively, within the 22.9-Mb syntenic region on Mmu16. We found that Dp(16Tiam1-Kcnj6)Yey/+, but not Dp(16)1Yey/Df(16Tiam1-Kcnj6)Yey, resulted in heart defects, indicating that triplication of the Tiam1-Knj6 region is necessary and sufficient to cause DS-associated heart defects. Our transcriptional analysis of Dp(16Tiam1-Kcnj6)Yey/+ embryos confirmed elevated expression levels for the genes located in the Tiam-Kcnj6 region. Therefore, we established the smallest critical genomic region for DS-associated heart defects to lay the foundation for identifying the causative gene(s) for this phenotype.
Sonic Hedgehog and its GLI transcriptional effectors control foliation complexity during cerebellar morphogenesis by promoting granule cell precursor proliferation. Here, we reveal a novel contribution of Sonic Hedgehog-GLI signaling to cerebellar patterning and cell differentiation by generating mice with targeted deletion of Suppressor of Fused (SuFu), a regulator of Sonic Hedgehog signaling, in the mid-hindbrain. Postnatal SuFu-deficient mice exhibit impaired motor coordination and severe cerebellar mispatterning. SuFu conditional knock-out embryos display abnormal mid-hindbrain morphology associated with misexpression of Fgf8, and delayed differentiation and abnormal migration of major cerebellar cell types. Sonic Hedgehog is ectopically expressed in the external granule layer and Hedgehog signaling is upregulated. While expression of full-length GLI transcriptional activators downstream of Hedgehogs is markedly reduced, a processed form of GLI3, a transcriptional repressor, is essentially lost. Genetic expression of a Gli3 allele encoding GLI3 repressor in SuFu-deficient mice largely rescues abnormal cerebellar patterning and cell differentiation observed in mice with SuFu deficiency alone. Together, our data demonstrate that SuFu controls cerebellar patterning and cell differentiation in a GLI3 repressor-dependent manner.
Neurocognitive assessment in individuals with intellectual disabilities requires a well-validated test battery. To meet this need, the Arizona Cognitive Test Battery (ACTB) has been developed specifically to assess the cognitive phenotype in Down syndrome (DS). The ACTB includes neuropsychological assessments chosen to 1) assess a range of skills, 2) be non-verbal so as to not confound the neuropsychological assessment with language demands, 3) have distributional properties appropriate for research studies to identify genetic modifiers of variation, 4) show sensitivity to within and between sample differences, 5) have specific correlates with brain function, and 6) be applicable to a wide age range and across contexts. The ACTB includes tests of general cognitive ability and prefrontal, hippocampal and cerebellar function. These tasks were drawn from the Cambridge Neuropsychological Testing Automated Battery (CANTAB) and other established paradigms. Alongside the cognitive testing battery we administered benchmark and parent-report assessments of cognition and behavior. Individuals with DS (n=74, ages 7-38 years) and mental age (MA) matched controls (n=50, ages 3-8 years) were tested across 3 sites. A subsample of these groups were used for between-group comparisons, including 55 individuals with DS and 36 mental age matched controls. The ACTB allows for low floor performance levels and participant loss. Floor effects were greater in younger children. Individuals with DS were impaired on a number ACTB tests in comparison to a MA-matched sample, with some areas of spared ability, particularly on tests requiring extensive motor coordination. Battery measures correlated with parent report of behavior and development. The ACTB provided consistent results across contexts, including home vs. lab visits, cross-site, and among individuals with a wide range of socio-economic backgrounds and differences in ethnicity. The ACTB will be useful in a range of outcome studies, including clinical trials and the identification of important genetic components of cognitive disability.
Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.
Down syndrome (DS) is caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is the most common genetic cause for developmental cognitive disability. The regions on Hsa21 are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this report, we describe a new mouse model for DS that carries duplications spanning the entire Hsa21 syntenic regions on all three mouse chromosomes. This mouse mutant exhibits DS-related neurological defects, including impaired cognitive behaviors, reduced hippocampal long-term potentiation and hydrocephalus. These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Because of its desirable genotype and phenotype, this mutant may have the potential to serve as one of the reference models for further understanding the developmental cognitive disability associated with DS and may also be used for developing novel therapeutic interventions for this clinical manifestation of the disorder. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Down syndrome (trisomy 21) is the most common cause of mental retardation in children and leads to marked deficits in contextual learning and memory. In rodents, these tasks require the hippocampus and are mediated by several inputs, particularly those originating in the locus coeruleus. These afferents mainly use norepinephrine as a transmitter. To explore the basis for contextual learning defects in Down syndrome, we examined the Ts65Dn mouse model. These mice, which have three copies of a fragment of mouse chromosome 16, exhibited significant deficits in contextual learning together with dysfunction and degeneration of locus coeruleus neurons. However, the postsynaptic targets of innervation remained responsive to noradrenergic receptor agonists. Indeed, despite advanced locus coeruleus degeneration, we were able to reverse contextual learning failure by using a prodrug for norepinephrine called l-threo-3,4-dihydroxyphenylserine, or xamoterol, a beta(1)-adrenergic receptor partial agonist. Moreover, an increased gene dosage of App, in the context of Down syndrome, was necessary for locus coeruleus degeneration. Our findings raise the possibility that restoring norepinephrine-mediated neurotransmission could reverse cognitive dysfunction in Down syndrome.
The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.
Hedgehog (Hh) signaling is crucial for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hedgehog-interacting protein (Hhip) is a surface receptor antagonist that is equipotent against all three mammalian Hh homologs. The crystal structures of human HHIP alone and bound to Sonic hedgehog (SHH) now reveal that HHIP is comprised of two EGF domains and a six-bladed beta-propeller domain. In the complex structure, a critical loop from HHIP binds the pseudo active site groove of SHH and directly coordinates its Zn2+ cation. Notably, sequence comparisons of this SHH binding loop with the Hh receptor Patched (Ptc1) ectodomains and HHIP- and PTC1-peptide binding studies suggest a 'patch for Patched' at the Shh pseudo active site; thus, we propose a role for Hhip as a structural decoy receptor for vertebrate Hh.
Sustained Sonic hedgehog (Shh) pathway activity is associated with tumorigenesis in a wide variety of tissues. Mutational inactivation of Shh receptor Patched (Ptch) and a downstream gene Suppressor of fused (Sufu), both of which are negative regulators of the pathway, increases susceptibility to cerebellum cancer in humans and mice. Sufu is a binding partner of Shh pathway transcription factor Gli. Recent data indicate that inactivation of Sufu, through either gene targeting in mice or RNAi-mediated silencing in cultured fibroblasts, is sufficient to turn on Shh target gene expression. Here, we report that Sufu is degraded rapidly in certain cancer cells and we show that Shh signaling promotes ubiquitination of Sufu, which leads to its destruction in the proteasomes. We identified an ubiquitin attachment site on K257 of Sufu, and showed that Sufu-K257R mutant is more potent as a transcription repressor and cell growth inhibitor because of increased stability. These results indicate that Shh signaling regulates Sufu activity by inducing its turnover via the ubiquitin-proteasome system.
Trisomy 21 or Down syndrome (DS) is the most frequent genetic cause of mental retardation, affecting one in 800 live born human beings. Mice with segmental trisomy 16 (Ts65Dn mice) are at dosage imbalance for genes corresponding to those on human chromosome 21q21-22.3--which includes the so-called DS 'critical region'. They do not show early-onset of Alzheimer disease pathology; however, Ts65Dn mice do demonstrate impaired performance in a complex learning task requiring the integration of visual and spatial information. The reproducibility of this phenotype among Ts65Dn mice indicates that dosage imbalance for a gene or genes in this region contributes to this impairment. The corresponding dosage imbalance for the human homologues of these genes may contribute to cognitive deficits in DS.
Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most common cause of congenital bowel obstruction with an incidence of 1 in 5000 live births. Recently, linkage of an incompletely penetrant, dominant form of HSCR was reported, followed by identification of mutations in the RET receptor tyrosine kinase. To determine the frequency of RET mutations in HSCR and correlate genotype with phenotype, we have screened for mutations among 80 HSCR probands representing a wide range of phenotypes and family structures. Polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis of RET's 20 exons for mutations among probands revealed eight putative mutations (10%). Sequence changes, which included missense, frameshift and complex mutations, were detected in both familial and isolated cases, among patients with both long- and short-segment HSCR and in three kindreds with other phenotypes (maternal deafness, talipes and malrotation of the gut, respectively). Two mutations (C609Y and C620R) we identified have previously been associated with multiple endocrine neoplasia type 2A (MEN2A), medullary thyroid carcinoma (MTC) and, on rare occasions, HSCR. Thus, while HSCR family members may be at risk for developing neuroendocrine tumors, it follows that identical mutations in RET may be able to participate in the pathogenesis of distinct phenotypes. Our data suggest that: (i) the overall frequency of RET mutations in HSCR patients is low and therefore, other genetic and/or environmental determinants contribute to the majority of HSCR susceptibility, and (ii) at present, there is no obvious relationship between RET genotype and HSCR phenotype.
The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.
Sonic hedgehog, Patched and Gli are components of a mammalian signalling pathway that has been conserved during evolution and which has a central role in the control of pattern formation and cellular proliferation during development. Here we identify the human Suppressor-of-Fused (SUFUH) complementary DNA and show that the gene product interacts physically with the transcriptional effector GLI-1, can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signalling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signalling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses.
Hedgehog (HH) signalling is involved in the development of numerous embryonic tissues. In humans,germline mutations in hedgehog pathway components cause congenital malformations and somatic mutations are associated with cancers. The basic framework of the HH pathway was elucidated in the fruitfly, Drosophila melanogaster, and this pathway is largely conserved in vertebrates, although some important differences have been noted. The current paradigm of the "canonical" pathway views HH signalling as a series of repressive interactions which culminates in GLI-mediated transcriptional regulation of a variety of cellular processes. Definitions of "non-canonical" signalling stem from examples where the response to HH morphogen deviates from this paradigm and, according to current reports, three general scenarios of noncanonical HH signalling can be defined: (1) Signalling that involves HH pathway components but which is independent of GLI-mediated transcription; (2) Direct interaction of HH signalling components with components of other molecular pathways; and (3) "Non-contiguous" or "atypical" interaction of core HH pathway components with one another. Currently, the evidence supporting non-canonical HH signalling is not conclusive. However, Sonic hedgehog (SHH) has been shown to regulate cell migration and axon guidance in several contexts, and some of these processes are independent of downstream components of the HH pathway, and presumably the transcriptional response to morphogen. Furthermore, biochemical studies have shown that the HH receptor, PTCH1, can directly interact both with Cyclin B1 and caspases, to inhibit cell proliferation and to promote apoptosis, respectively, and that these functions are inhibited in the presence of morphogen. Genetic analysis of orthologues of the HH pathway in nematode worms further supports the notion that PTCH1-related molecules can function independently of other components of the canonical HH pathway, and the phenotypes of mice with point mutations in the Ptch1 gene offer clues as to the processes that non-canonical HH signalling might regulate. While none of these evidences are conclusive,collectively they point to the existence of added complexity in the HH pathway in the form of non-canonical pathways. A major difficulty in studying this problem is that canonical and non-canonical pathways are likely to act in parallel, and so in many situations it will not be possible to implicate non-canonical responses in certain cellular processes simply by excluding a role for the canonical pathway-directed analyses of non-canonical HH signalling are therefore necessary. The aim of this review is to present the cumulative evidence supporting non-canonical HH signalling, with the hope of promoting further enquiry into this area.
The presence of an additional copy of HSA21 chromosome in Down syndrome (DS) individuals leads to the overexpression of 30-50% of HSA21 genes. This upregulation can, in turn, trigger a deregulation on the expression of non-HSA21 genes. Moreover, the overdose of HSA21 microRNAs (miRNAs) may result in the downregulation of its target genes. Additional complexity can also arise from epigenetic changes modulating gene expression. Thus, a myriad of transcriptional and posttranscriptional alterations participate to produce abnormal phenotypes in almost all tissues and organs of DS individuals. The study of the physiological roles of genes dysregulated in DS, as well as their characterization in murine models with gene(s) dosage imbalance, pointed out several genes, and functional noncoding elements to be particularly critical in the etiology of DS. Recent findings indicate that gene therapy strategies-based on the introduction of genetic elements by means of delivery vectors-toward the correction of phenotypic abnormalities in DS are also very promising tool to identify HSA21 and non-HSA21 gene candidates, contributing to DS phenotype. In this chapter, we focus on the impact of normalizing the expression levels of up or downregulated genes to rescue particular phenotypes of DS. Attempts toward gene-based treatment approaches in mouse models will be discussed as new opportunities to ameliorate DS alterations.
Mouse models are key elements to better understand the genotype-phenotype relationship and the physiopathology of Down syndrome (DS). Even though the mouse will never recapitulate the whole spectrum of intellectual disabilities observed in the DS, mouse models have been developed over the recent decades and have been used extensively to identify homologous genes or entire regions homologous to the human chromosome 21 (Hsa21) that are necessary or sufficient to induce DS cognitive features. In this chapter, we review the principal mouse DS models which have been selected and engineered over the years either for large genomic regions or for a few or a single gene of interest. Their analyses highlight the complexity of the genetic interactions that are involved in DS cognitive phenotypes and also strengthen the hypothesis on the multigenic nature of DS. This review also addresses future research challenges relative to the making of new models and their combination to go further in the characterization of candidates and modifier of the DS features.
The study of cognitive function in Down syndrome (DS) has advanced rapidly in the past decade. Mouse models have generated data regarding the neurological basis for the specific cognitive profile of DS (i.e., deficits in aspects of hippocampal, prefrontal, and cerebellar function) and have uncovered pharmacological treatments with the potential to affect this phenotype. Given this progress, the field is at a juncture in which we require assessments that may effectively translate the findings acquired in mouse models to humans with DS. In this chapter, we describe the cognitive profile of humans with DS and associated mouse models, discussing the ways in which we may merge these findings so as to more fully understand cognitive strengths and weaknesses in this population. New directions for approaches to cognitive assessment in mice and humans are discussed.
Major efforts in Down syndrome (DS) research have been directed at the identification and functional characterization of genes encoded by human chromosome 21 (HSA21). In parallel with this, tissue samples and cell lines derived from individuals with DS have been examined for abnormalities in gene expression and cellular morphology, and mouse models of DS have been characterized for abnormalities at the molecular, cellular, electrophysiological, and behavioral level. One goal of such investigations has been the identification of effective targets for pharmacotherapies that can prevent or correct the abnormalities and, by extension to human clinical trials, prevent or lessen aspects of the cognitive deficits seen in people with DS. Because it is caused by an extra copy of an entire chromosome, DS has been considered by some as too complicated a genetic perturbation to be amenable to postnatal pharmacological interventions. However, recent data from experiments with one mouse model, the Ts65Dn, have clearly demonstrated that several pharmacological interventions can indeed rescue DS-relevant learning and memory deficits. Extension of mouse data to successful human clinical trials will be aided by understanding the molecular basis of successful drug treatments, that is, how increased expression of HSA21 genes perturbs molecular mechanisms that are targeted and rescued by specific drugs. Here, we review information on HSA21 genes, their expression and their likely contributions to the DS phenotype. We then describe results of a bioinformatics effort that integrates information on genes known to cause intellectual disability when mutated, the pathways in which these genes function, and how these pathways are impacted by HSA21 encoded proteins. This pathway approach to the molecular basis of ID in DS aids in understanding why some drug therapies have been successful in the Ts65Dn and in predicting whether these same drugs are likely to be successful in treating ID in DS. These data can be used to design new experiments and interpret information for prediction of additional targets for effective drug treatments.
Clinical trials with drugs aimed at treatment of Alzheimer disease to decelerate cognitive decline and translated mimetically to demented and young nondemented Down syndrome patients have been unable to demonstrate improvements in cognitive performance and functioning. Unfortunately, results from clinical trials do not support the use of NMDA antagonists like memantine and we should await at the development of safer GABA(A) antagonists to conclude about the efficacy of approaching Down syndrome therapeutics by modulating neurotransmission systems altered in this pathology. The use of folinic acid or antioxidants in DS patients is not supported by scientific evidence and do not provide improvement in cognitive performance to patients. Alternatively to the modulation of neurotransmission systems, future therapeutic approaches should focus at normalizing the expression levels or function of candidate molecules. Epigallocatechin gallate, a green tea polyphenol, that modulates DYRK1A functioning has already shown preliminarily that this approach may prove useful in therapeutics.
Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS). The finished human genome sequence provides a thorough catalog of the genetic elements whose altered dosage perturbs development and function in DS. However, understanding how small alterations in the steady state transcript levels for <2% of human genes can disrupt development and function of essentially every cell presents a more complicated problem. Mouse models that recapitulate specific aspects of DS have been used to identify changes in brain morphogenesis and function. Here we provide a few examples of how trisomy for specific genes affects the development of the cortex and cerebellum to illustrate how gene dosage effects might contribute to divergence between the trisomic and euploid brains.
Mental retardation in Down syndrome (DS) appears to be related to severe neurogenesis impairment during critical phases of brain development. Recent lines of evidence in the cerebellum of a mouse model for DS (the Ts65Dn mouse) have shown a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting involvement of the Shh pathway in the neurogenesis defects of DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. By using an in vitro model of NPCs obtained from the subventricular zone and hippocampus, we found that trisomic NPCs had an increased expression of the Shh receptor Patched1 (Ptch1), a membrane protein that suppresses the action of a second receptor, Smoothened (Smo), thereby maintaining the pathway in a repressed state. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. The overexpression of Ptch1 in trisomic NPCs resulted from increased levels of AICD [a transcription-promoting fragment of amyloid precursor protein (APP)] and increased AICD binding to the Ptch1 promoter. Our data provide novel evidence that Ptch1 overexpression underlies derangement of the Shh pathway in trisomic NPCs with consequent proliferation impairment. The demonstration that Ptch1 overexpression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.
Evidence in mouse models for Down syndrome (DS) and human fetuses with DS clearly shows severe neurogenesis impairment in various telencephalic regions, suggesting that this defect may underlie the cognitive abnormalities of DS. As cerebellar hypotrophy and motor disturbances are part of the clinical features of DS, the goal of our study was to establish whether these defects may be related to neurogenesis impairment during cerebellar development. We found that in fetuses with DS (17-21 weeks of gestation) the cerebellum had an immature pattern, a reduced volume and notably fewer cells (-25%/-50%) in all cerebellar layers. Immunohistochemistry for Ki-67, a marker of cycling cells, showed impaired proliferation (-17%/-50%) of precursors from both cerebellar neurogenic regions (external granular layer and ventricular zone). No differences in apoptotic cell death were found in DS vs. control fetuses. The current study provides novel evidence that in the cerebellum of DS fetuses there is a generalized hypocellularity and that this defect is due to proliferation impairment, rather than to an increased cell death. The reduced proliferation potency found in the DS fetal cerebellum, in conjunction with previous evidence, strengthens the idea that the trisomic brain is characterized by widespread neurogenesis disruption.
Hirschsprung's disease (HSCR) is characterized by absence of the enteric nervous system in a variable portion of the distal gut. Affected infants usually present in the days after birth with bowel obstruction. Despite surgical advances, long-term outcomes remain variable. In the last 2 decades, great advances have been made in understanding the genes and molecular biological mechanisms that underlie the disease. In addition, our understanding of normal enteric nervous system development and how motility develops in the developing fetus and infant has also increased. This review aims to draw these strands together to explain the developmental and biological basis of HSCR, and how this knowledge may be used in the future to aid children with HSCR.
Improvements in medical interventions for people with Down's syndrome have led to a substantial increase in their longevity. Diagnosis and treatment of neurological complications are important in maintaining optimal cognitive functioning. The cognitive phenotype in Down's syndrome is characterised by impairments in morphosyntax, verbal short-term memory, and explicit long-term memory. However, visuospatial short-term memory, associative learning, and implicit long-term memory functions are preserved. Seizures are associated with cognitive decline and seem to cause additional decline in cognitive functioning, particularly in people with Down's syndrome and comorbid disorders such as autism. Vision and hearing disorders as well as hypothyroidism can negatively impact cognitive functioning in people with Down's syndrome. Dementia that resembles Alzheimer's disease is common in adults with Down's syndrome. Early-onset dementia in adults with Down's syndrome does not seem to be associated with atherosclerotic complications.
The Hedgehog signalling pathway controls numerous developmental processes. In response to Hedgehog, Smoothened (Smo), a seven-pass transmembrane protein, orchestrates pathway signalling and controls transcription factor activation. In the absence of Hedgehog, the receptor Patched indirectly inhibits Smo in a catalytic manner. Many questions surrounding Smo activation and signalling remain. Recent findings in Drosophila and vertebrate systems have provided strong evidence that Smo acts as a G-protein-coupled receptor. We discuss the role and regulation of Smo and reassess similarities between Smo and G-protein-coupled receptors. We also examine recently identified members of the invertebrate and vertebrate Smo signalling cascades that are typical components of G-protein-coupled receptor pathways. Greater understanding of the mechanisms of Smo activation and its signalling pathways will allow implementation of novel strategies to target disorders related to disruption of Hh signalling.
Although de la Cruz and colleagues showed as early as 1977 that the outflow tract was added after the heart tube formed, the source of these secondarily added cells was not identified for nearly 25 years. In 2001, three pivotal publications described a secondary or anterior heart field that contributed to the developing outflow tract. This review details the history of the heart field, the discovery and continuing elucidation of the secondarily adding myocardial cells, and how the different populations identified in 2001 are related to the more recent lineage tracing studies that defined the first and second myocardial heart fields/lineages. Much recent work has focused on secondary heart field progenitors that give rise to the myocardium and smooth muscle at the definitive arterial pole. These progenitors are the last to be added to the arterial pole and are particularly susceptible to abnormal development, leading to conotruncal malformations in children. The major signaling pathways (Wnt, BMP, FGF8, Notch, and Shh) that control various aspects of secondary heart field progenitor behavior are discussed.
Aneuploidies are common chromosomal defects that result in growth and developmental deficits and high levels of lethality in humans. To gain insight into the biology of aneuploidies, we manipulated mouse embryonic stem cells and generated a trans-species aneuploid mouse line that stably transmits a freely segregating, almost complete human chromosome 21 (Hsa21). This "transchromosomic" mouse line, Tc1, is a model of trisomy 21, which manifests as Down syndrome (DS) in humans, and has phenotypic alterations in behavior, synaptic plasticity, cerebellar neuronal number, heart development, and mandible size that relate to human DS. Transchromosomic mouse lines such as Tc1 may represent useful genetic tools for dissecting other human aneuploidies.
Hedgehog (HH) signalling is involved in the development of numerous embryonic tissues. In humans, germline mutations in hedgehog pathway components cause congenital malformations and somatic mutations are associated with cancers. The basic framework of the HH pathway was elucidated in the fruit fly, Drosophila melanogaster, and this pathway is largely conserved in vertebrates, although some important differences have been noted. The current paradigm of the 'canonical' pathway views HH signalling as a series of repressive interactions which culminates in GLI-mediated transcriptional regulation of a variety of cellular processes. Definitions of 'non-canonical' signalling stem from examples where the response to HH morphogen deviates from this paradigm and, according to current reports, three general scenarios of non-canonical HH signalling can be defined: (1) Signalling that involves HH pathway components but which is independent of GLI-mediated transcription; (2) Direct interaction of HH signalling components with components of other molecular pathways; and (3) 'Non-contiguous' or 'atypical' interaction of core HH pathway components with one another. Currently, the evidence supporting non-canonical HH signalling is not conclusive. However, Sonic hedgehog (SHH) has been shown to regulate cell migration and axon guidance in several contexts, and some of these processes are independent of downstream components of the HH pathway, and presumably the transcriptional response to morphogen. Furthermore, biochemical studies have shown that the HH receptor, PTCH1, can directly interact both with Cyclin B1 and caspases, to inhibit cell proliferation and to promote apoptosis, respectively, and that these functions are inhibited in the presence of morphogen. Genetic analysis of orthologues of the HH pathway in nematode worms further supports the notion that PTCH1-related molecules can function independently of other components of the canonical HH pathway, and the phenotypes of mice with point mutations in the Ptch1 gene offer clues as to the processes that non-canonical HH signalling might regulate. While none of these evidences are conclusive, collectively they point to the existence of added complexity in the HH pathway in the form of non-canonical pathways. A major difficulty in studying this problem is that canonical and non-canonical pathways are likely to act in parallel, and so in many situations it will not be possible to implicate non-canonical responses in certain cellular processes simply by excluding a role for the canonical pathway -- directed analyses of non-canonical HH signalling are therefore necessary. The aim of this review is to present the cumulative evidence supporting non-canonical HH signalling, with the hope of promoting further enquiry into this area.
Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left-right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant-negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.
SCUBE2 [signal peptide, CUB domain, EGF (epidermal growth factor)-like protein 2] belongs to an evolutionarily conserved SCUBE protein family, which possesses domain organization characteristic of an N-terminal signal peptide sequence followed by nine EGF-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C-terminus. Despite several genetic analyses suggesting that the zebrafish orthologue of the mammalian SCUBE2 gene participates in HH (Hedgehog) signalling, the complete full-length cDNA and biochemical function for mammalian SCUBE2 on HH signalling remains uninvestigated. In the present study, we isolated the full-length cDNA and studied the role of human SCUBE2 in the HH signalling cascade. When overexpressed, recombinant human SCUBE2 manifests as a secreted surface-anchored glycoprotein. Deletion mapping analysis defines the critical role of the spacer region and/or cysteine-rich repeats for membrane association. Further biochemical analyses and functional reporter assays demonstrated that human SCUBE2 can specifically interact with SHH (Sonic Hedgehog) and SHH receptor PTCH1 (Patched-1), and enhance the SHH signalling activity within the cholesterol-rich raft microdomains of the plasma membranes. Together, our results reveal that human SCUBE2 is a novel positive component of the HH signal, acting upstream of ligand binding at the plasma membrane. Thus human SCUBE2 could play important roles in HH-related biology and pathology, such as during organ development and tumour progression.
The genesis of the septal structures of the mammalian heart is central to understanding the ontogeny of congenital heart disease and the evolution of cardiac organogenesis. We found that Hedgehog (Hh) signaling marked a subset of cardiac progenitors specific to the atrial septum and the pulmonary trunk in the mouse. Using genetic inducible fate mapping with Gli1(CreERT2), we marked Hh-receiving progenitors in anterior and posterior second heart field splanchnic mesoderm between E8 and E10. In the inflow tract, Hh-receiving progenitors migrated from the posterior second heart field through the dorsal mesocardium to form the atrial septum, including both the primary atrial septum and dorsal mesenchymal protrusion (DMP). In the outflow tract, Hh-receiving progenitors migrated from the anterior second heart field to populate the pulmonary trunk. Abrogation of Hh signaling during atrial septal progenitor specification resulted in atrial and atrioventricular septal defects and hypoplasia of the developing DMP. Hedgehog signaling appeared necessary and sufficient for atrial septal progenitor fate: Hh-receiving cells rendered unresponsive to the Hh ligand migrated into the atrium in normal numbers but populated the atrial free wall rather than the atrial septum. Conversely, constitutive activation of Hh signaling caused inappropriate enlargement of the atrial septum. The close proximity of posterior second heart field cardiac progenitors to pulmonary endoderm suggested a pulmonary source for the Hh ligand. We found that Shh is required in the pulmonary endoderm for atrial septation. Therefore, Hh signaling from distinct pulmonary and pharyngeal endoderm is required for inflow and outflow septation, respectively. These data suggest a model in which respiratory endoderm patterns the morphogenesis of cardiac structural components required for efficient cardiopulmonary circulation.
Individuals with Down syndrome (DS) display a 40-fold greater risk of Hirschsprung disease (HSCR) than the general population of newborns implicating chromosome 21 in HSCR etiology. Here we demonstrate that the RET enhancer polymorphism RET+9.7 (rs2435357:C>T) at chromosome 10q11.2 is associated with HSCR in DS individuals both by transmission disequilibrium (P=0.0015) and case-control (P=0.0115) analysis of matched cases. Interestingly, the RET+9.7 T allele frequency is significantly different between individuals with DS alone (0.26+/-0.04), HSCR alone (0.61+/-0.04), and those with HSCR and DS (0.41+/-0.04), demonstrating an association and interaction between RET and chromosome 21 gene dosage. This is the first report of a genetic interaction between a common functional variant (rs2435357) and a not infrequent copy number error (chromosome 21 dosage) in two human developmental disorders.
The hedgehog and mitogen-activated protein kinase (MAPK) signaling pathways regulate growth in many tumors, suggesting cooperation between these two pathways in the regulation of cell proliferation. However, interactions between these pathways have not been extensively studied. We assessed cross-talk between hedgehog and MAPK signaling in the regulation of cell proliferation in gastric cancer. We showed that PTCH expression was significantly correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P = 0.016) as well as SHH expression (P = 0.034) in the 35 gastric cancers assessed by immunohistochemistry. Indeed, MAPK signaling increased the GLI transcriptional activity and induced the expression of hedgehog target genes in gastric cancer cells. The inductive effect of activated KRAS and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1 was blocked by the suppressor of fused (SUFU), indicating that MAPK signaling regulates GLI activity via a SUFU-independent process. Moreover, the deletion of the NH2-terminal domain of GLI1 gene resulted in reduced response to MEK1 stimulation. Our results suggest that the KRAS-MEK-ERK cascade has a positive regulatory role in GLI transcriptional activity in gastric cancer.
Trisomy 21 results in phenotypes collectively referred to as Down syndrome (DS) including characteristic facial dysmorphology. Ts65Dn mice are trisomic for orthologs of about half of the genes found on human chromosome 21 and exhibit DS-like craniofacial abnormalities, including a small dysmorphic mandible. Quantitative analysis of neural crest (NC) progenitors of the mandible revealed a paucity of NC and a smaller first pharyngeal arch (PA1) in Ts65Dn as compared to euploid embryos. Similar effects in PA2 suggest that trisomy causes a neurocristopathy in Ts65Dn mice (and by extension, DS). Further analyses demonstrated deficits in delamination, migration, and mitosis of trisomic NC. Addition of Sonic hedgehog (Shh) growth factor to trisomic cells from PA1 increased cell number to the same level as untreated control cells. Combined with previous demonstrations of a deficit in mitogenic response to Shh by trisomic cerebellar granule cell precursors, these results implicate common cellular and molecular bases of multiple DS phenotypes.
The Baltimore-Washington Infant Study is a population-based case-control study that seeks to identify risk factors for cardiovascular malformations. Between 1981 and 1986, a total of 2102 infants with cardiovascular malformations were ascertained, among whom 271 (12.9%) also had a chromosome abnormality. Among 2328 random control subjects, only two had a chromosome abnormality. Down syndrome with cardiovascular malformations had a maternal age-adjusted regional prevalence of 4.33/10,000 for the white population and 3.70/10,000 for the nonwhite population. Endocardial cushion defect, the predominant cardiac abnormality in Down syndrome (60.1%), rarely occurred as an isolated cardiac lesion (2.8%). The absence of transpositions and the rarity of heterotaxias and of right- and left-sided obstructive lesions in trisomies indicate that there may be a genetic influence on specific embryologic mechanisms. Alimentary tract lesions were more common in Down syndrome than among euploid patients with heart disease and more severe than in control subjects. Urinary tract lesions also occurred in excess of the rate in control subjects. The coexistence of these major malformations with heart disease raises the possibility of incomplete expression of the VA(C)TER (vertebral, anal, cardiac, tracheal, esophageal renal) association. The selective association of chromosome abnormalities with certain cardiovascular defects is now beginning to be explained by reported embryologic studies on cellular characteristics. An explanation of the negative association with transposition and obstructive lesions requires further multidisciplinary studies on genetic and epigenetic factors.
Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.
The protein Sonic hedgehog (Shh) is essential for a variety of patterning events during development. It is the signal from the notochord that induces ventral cell fate in the neural tube and somites, and is the polarizing signal for patterning of the anterior-posterior axis of the developing limb bud. Because of these and other inductive functions of Shh, it is important to understand how the Hedgehog (Hh) signal is received by the target cells. Here we describe binding studies using labelled Shh that strongly suggest that the Hh receptor is encoded by patched (ptc), a gene first identified in genetic screens in Drosophila.
The PATCHED (PTC) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome
(BCNS). Functions ofPTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation died during embryogenesis and were found to have open and overgrown neural tubes. Two Shh target genes, ptc itself and Gli, were derepressed in the ectoderm and mesoderm but not in the endoderm. Shh targets that are, under normal conditions, transcribed
ventrally were aberrantly expressed in dorsal and lateral neural tube cells. Thus Ptc appears to be essential for repression
of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation were larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas, abnormalities
also seen in BCNS patients.
Ts65Dn mouse displays a partial triplication of chromosome 16 and is adopted as a model for Down syndrome (DS). It is known that Ts65Dn mice present memory deficiencies. In order to gain insight into the cause of these deficiencies, we studied the possibility of changes in volumes and neuronal numbers in different regions of the hippocampus (dentate gyrus, CA3, CA2 and CA1) in trisomic mice as compared to control littermates using stereological methods. The mean hippocampal volumes of Ts65Dn mice did not show significant differences as compared to controls, except in CA2 where there was a barely significant decrease. However, mean neuron number was significantly lower in Ts65Dn mice than in controls in dentate gyrus (43.7 x 10(4), CV 21%, n = 5, vs. 30.4 x 10(4), CV 18.1%, n = 4) and higher in CA3 (23.1 x 10(4), CV 18.9% vs. 33.3 x 10(4), CV 14.9%). These quantitative changes may account for the memory deficiencies observed in Ts65Dn mice.
Cerebellar granule cells are the most abundant type of neuron in the brain, but the molecular mechanisms that control their generation are incompletely understood. We show that Sonic hedgehog (Shh), which is made by Purkinje cells, regulates the division of granule cell precursors (GCPs). Treatment of GCPs with Shh prevents differentiation and induces a potent, long-lasting proliferative response. This response can be inhibited by basic fibroblast growth factor or by activation of protein kinase A. Blocking Shh function in vivo dramatically reduces GCP proliferation. These findings provide insight into the mechanisms of normal growth and tumorigenesis in the cerebellum.
It used to be thought that the atrioventricular septum was predominantly the product of the atrioventricular endocardial cushions. In a previous study, we have shown that multiple developmental primordia are of importance in its formation. With this in mind, we have evaluated cardiac morphogenesis in the mouse with trisomy 16, an animal model with a high incidence of atrioventricular septal defects. Normal and trisomic fetuses from an Rb(11.16)2H/Rb(16.17)7Bnr x C57BL/6J cross were collected on days 10 to 15 of gestation and examined by scanning electron microscopy and histological serial sectioning. No evidence was found to suggest that atrioventricular septal defect could be explained simply on the basis of "failure of fusion" between the atrioventricular endocardial cushions. Rather, our findings supported two other developmental elements as being important in the genesis of atrioventricular septal defect. The first is an alteration in the configuration of the heart tube, with inadequate remodeling of the inner heart curvature. This resulted in the failure of the atrioventricular junction to expand to the right, with subsequent malalignment of the atrioventricular endocardial cushions with the proximal outflow cushions. The second is a variability in the connection of the primary atrial cardiac segment to the body of the embryo, the so-called dorsal mesocardium, which influences its relationship to the extracardiac mediastinal mesoderm. There appeared little difference in the connection between normal and trisomic embryos at the stage of 20 to 25 somites, but the area subsequently showed marked changes. In most trisomic embryos, the connection with the mediastinal mesoderm of the body was over a larger area than seen in normal embryos. As this area of attachment encloses the pulmonary pit, the entry point of the pulmonary vein, this gives potential for variation in the connection of the pulmonary vein. In addition, in the majority of trisomic embryos, the right pulmonary ridge (the spina vestibuli) did not accumulate extracardiac mesoderm, nor did it undergo the pronounced forward growth seen in normal embryos of equivalent stages. Consequently, the trisomic embryos show incomplete formation of both the atrial and the atrioventricular septal structures.
Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input - that from the inferior olive via climbing fibres and that from the granule neurons via parallel fibres. The precursors of granule neurons proliferate at the surface of the developing cerebellumin the external granule layer (EGL), which persists until postnatal day 14 in the mouse [1]. PCs are thought to provide trophic support for granule neurons [2][3] and to stimulate the proliferation of cells in the EGL [4], but the signalling molecules that mediate these cell-cell interactions have not been identified. I show here that PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and that dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes of which expression is upregulated in response to Hedgehog signalling (see [5] and references therein). Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies [6] disrupted cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulated BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL.
The molecular bases of brain development and CNS malignancies remain poorly understood. Here we show that Sonic hedgehog (Shh) signaling controls the development of the cerebellum at multiple levels. SHH is produced by Purkinje neurons, it is required for the proliferation of granule neuron precursors and it induces the differentiation of Bergmann glia. Blocking SHH function in vivo results in deficient granule neuron and Bergmann glia differentiation as well as in abnormal Purkinje neuron development. Thus, our findings provide a molecular model for the growth and patterning of the cerebellum by SHH through the coordination of the development of cortical cerebellar cell types. In addition, they provide a cellular context for medulloblastomas, childhood cancers of the cerebellum.
Sonic hedgehog (Shh) is well known for its role in patterning tissues, including structures of the head. Haploinsufficiency for SHH in humans results in holoprosencephaly, a syndrome characterized by facial and forebrain abnormalities. Shh null mice have cyclopia and loss of branchial arch structures. It is unclear, however, whether these phenotypes arise solely from the early function of Shh in patterning midline structures, or whether Shh plays other roles in head development.
To address the role of Shh after floorplate induction, we inhibited Shh signaling by injecting hybridoma cells that secrete a function-blocking anti-Shh antibody into the chick cranial mesenchyme. The antibody subsequently bound to Shh in the floorplate, notochord, and the pharyngeal endoderm. Perturbation of Shh signaling at this stage resulted in a significant reduction in head size after 1 day, loss of branchial arch structures after 2 days, and embryos with smaller heads after 7 days. Cell death was significantly increased in the neural tube and neural crest after 1 day, and neural crest cell death was not secondary to the loss of neural tube cells.
Reduction of Shh signaling after neural tube closure resulted in a transient decrease in neural tube cell proliferation and an extensive increase in cell death in the neural tube and neural crest, which in turn resulted in decreased head size. The phenotypes observed after reduction of Shh are similar to those observed after cranial neural crest ablation. Thus, our results demonstrate a role for Shh in coordinating the proliferation and survival of cells of the neural tube and cranial neural crest.
Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. We demonstrate that PKA-dependent processing of vertebrate Gli3 in developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation.