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In JoVE (1)
Other Publications (10)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Cancer Cell
- Development (Cambridge, England)
- Current Topics in Developmental Biology
- Development (Cambridge, England)
- Development (Cambridge, England)
- Development (Cambridge, England)
- PloS One
- Neural Development
- Neural Development
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Articles by Sandra Blaess in JoVE
JoVE Neuroscience
Embriyonik ventral orta beyin Organotipik Dilim Kültürler: dopaminerjik Nöronal Gelişim Eğitim Bir Sistem In vitro
Institute of Reconstructive Neurobiology, University of Bonn
E12.5 fare embriyonik Ortabeyin Organotipik dilimler oluşturmak için bir yöntem açıklanmıştır. Organotipik dilim kültürleri dopaminerjik nöronlar veya diğer ventral orta beyin nöronları davranışını gözlemlemek için kullanılabilir.
Other articles by Sandra Blaess on PubMed
Beta1-integrins Are Critical for Cerebellar Granule Cell Precursor Proliferation
We have previously shown that mice with a CNS restricted knock-out of the integrin beta1 subunit gene (Itgb1-CNSko mice) have defects in the formation of lamina and folia in the cerebral and cerebellar cortices that are caused by disruption of the cortical marginal zones. Cortical structures in postnatal and adult Itgb1-CNSko animals are also reduced in size, but the mechanism that causes the size defect has remained unclear. We now demonstrate that proliferation of granule cell precursors (GCPs) is severely affected in the developing cerebellum of Itgb1-CNSko mice. In the absence of beta1 expression, GCPs lose contact with laminin in the meningeal basement membrane, cease proliferating, and differentiate prematurely. In vitro studies provide evidence that beta1 integrins act at least in part cell autonomously in GCPs to regulate their proliferation. Previous studies have shown that sonic hedgehog (Shh)-induced GCP proliferation is potentiated by the integrin ligand laminin. We show that Shh directly binds to laminin and that laminin-Shh induced cell proliferation is dependent on beta1 integrin expression in GCPs. Taken together, these data are consistent with a model in which beta1 integrin expression in GCPs is required to recruit a laminin-Shh complex to the surface of GCPs and to subsequently modulate the activity of signaling pathways that regulate proliferation.
Targeted Disruption of Beta1-integrin in a Transgenic Mouse Model of Human Breast Cancer Reveals an Essential Role in Mammary Tumor Induction
Despite evidence demonstrating the role of beta1-integrin in the regulation of cancer cell proliferation in vitro, the importance of this cell adhesion receptor during the initiation and progression of epithelial tumors in vivo remains unclear. Here we have used the Cre/LoxP1 recombination system to disrupt beta1-integrin function in the mammary epithelium of a transgenic mouse model of human breast cancer. Using this approach, we show that beta1-integrin expression is critical for the initiation of mammary tumorigenesis in vivo, and for maintaining the proliferative capacity of late-stage tumor cells. These observations provide a direct demonstration that beta1-integrin plays a critical role in both the initiation and maintenance of mammary tumor growth in vivo.
Spatial Pattern of Sonic Hedgehog Signaling Through Gli Genes During Cerebellum Development
The cerebellum consists of a highly organized set of folia that are largely generated postnatally during expansion of the granule cell precursor (GCP) pool. Since the secreted factor sonic hedgehog (Shh) is expressed in Purkinje cells and functions as a GCP mitogen in vitro, it is possible that Shh influences foliation during cerebellum development by regulating the position and/or size of lobes. We studied how Shh and its transcriptional mediators, the Gli proteins, regulate GCP proliferation in vivo, and tested whether they influence foliation. We demonstrate that Shh expression correlates spatially and temporally with foliation. Expression of the Shh target gene Gli1 is also highest in the anterior medial cerebellum, but is restricted to proliferating GCPs and Bergmann glia. By contrast, Gli2 is expressed uniformly in all cells in the developing cerebellum except Purkinje cells and Gli3 is broadly expressed along the anteroposterior axis. Whereas Gli mutants have a normal cerebellum, Gli2 mutants have greatly reduced foliation at birth and a decrease in GCPs. In a complementary study using transgenic mice, we show that overexpressing Shh in the normal domain does not grossly alter the basic foliation pattern, but does lead to prolonged proliferation of GCPs and an increase in the overall size of the cerebellum. Taken together, these studies demonstrate that positive Shh signaling through Gli2 is required to generate a sufficient number of GCPs for proper lobe growth.
Classical Embryological Studies and Modern Genetic Analysis of Midbrain and Cerebellum Development
The brain is a remarkably complex anatomical structure that contains a diverse array of subdivisions, cell types, and synaptic connections. It is equally extraordinary in its physiological properties, as it constantly evaluates and integrates external stimuli as well as controls a complicated internal environment. The brain can be divided into three primary broad regions: the forebrain, midbrain (Mb), and hindbrain (Hb), each of which contain further subdivisions. The regions considered in this chapter are the Mb and most-anterior Hb (Mb/aHb), which are derived from the mesencephalon (mes) and rhombomere 1 (r1), respectively. The dorsal Mb consists of the laminated superior colliculus and the globular inferior colliculus (Fig. 1A and B), which modulate visual and auditory stimuli, respectively. The dorsal component of the aHb is the highly foliated cerebellum (Cb), which is primarily attributed to controlling motor skills (Fig. 1A and B). In contrast, the ventral Mb/aHb (Fig. 1B) consists of distinct clusters of neurons that together comprise a network of nuclei and projections-notably, the Mb dopaminergic and Hb serotonergic and Mb/aHb cholinergic neurons (Fig. 1G and H), which modulate a collection of behaviors, including movement, arousal, feeding, wakefulness, and emotion. Historically, the dorsal Mb and Cb have been studied using the chick as a model system because of the ease of performing both cell labeling and tissue transplants in the embryo in ovo; currently DNA electroporation techniques are also used. More recently the mouse has emerged as a powerful genetic system with numerous advantages to study events underpinning Mb/aHb development. There is a diverse array of spontaneous mutants with both Mb- and Cb-related phenotypes. In addition, numerous gene functions have been enumerated in mouse, gene expression is similar across vertebrates, and powerful genetic tools have been developed. Finally, additional insight into Mb/aHb function has been gained from studies of genetic diseases, such as Parkinson's disease, schizophrenia, cancer, and Dandy Walker syndrome, that afflict the Mb/aHb in humans and have genetic counterparts in mouse. Accordingly, this chapter discusses a spectrum of experiments, including classic embryology, in vitro assays, sophisticated genetic methods, and human diseases. We begin with an overview of Mb and aHb anatomy and physiology and mes/r1 gene expression patterns. We then provide a summary of fate-mapping studies that collectively demonstrate the complex cell behaviors that occur while the Mb and aHb primordia are established during embryogenesis and discuss the integration of both anterior-posterior (A-P) and dorsal-ventral (D-V) patterning. Finally, we describe some aspects of postnatal development and some of the insights gained from human diseases.
The Level of Sonic Hedgehog Signaling Regulates the Complexity of Cerebellar Foliation
Foliation of the mouse cerebellum occurs primarily during the first 2 weeks after birth and is accompanied by tremendous proliferation of granule cell precursors (GCPs). We have previously shown that sonic hedgehog (Shh) signaling correlates spatially and temporally with fissure formation, and that Gli2 is the main activator driving Shh induced proliferation of embryonic GCPs. Here, we have tested whether the level of Shh signaling regulates the extent of cerebellar foliation. By progressively lowering signaling by removing Gli1 and Gli2 or the Shh receptor smoothened, we found the extent of foliation is gradually reduced, and that this correlates with a decrease in the duration of GCP proliferation. Importantly, the pattern of the remaining fissures in the mutants corresponds to the first fissures that form during normal development. In a complementary manner, an increase in the level and length of Shh signaling results in formation of an extra fissure in a position conserved in rat. The complexity of cerebellar foliation varies greatly between vertebrate species. Our studies have uncovered a mechanism by which the level and length of Shh signaling could be integral to determining the distinct number of fissures in each species.
Sonic Hedgehog Regulates Gli Activator and Repressor Functions with Spatial and Temporal Precision in the Mid/hindbrain Region
The midbrain and anterior hindbrain offer an ideal system in which to study the coordination of tissue growth and patterning in three dimensions. Two organizers that control anteroposterior (AP) and dorsoventral (DV) development are known, and the regulation of AP patterning by Fgf8 has been studied in detail. Much less is known about the mechanisms that control mid/hindbrain development along the DV axis. Using a conditional mutagenesis approach, we have determined how the ventrally expressed morphogen sonic hedgehog (Shh) directs mid/hindbrain development over time and space through positive regulation of the Gli activators (GliA) and inhibition of the Gli3 repressor (Gli3R). We have discovered that Gli2A-mediated Shh signaling sequentially induces ventral neurons along the medial to lateral axis, and only before midgestation. Unlike in the spinal cord, Shh signaling plays a major role in patterning of dorsal structures (tectum and cerebellum). This function of Shh signaling involves inhibition of Gli3R and continues after midgestation. Gli3R levels also regulate overall growth of the mid/hindbrain region, and this largely involves the suppression of cell death. Furthermore, inhibition of Gli3R by Shh signaling is required to sustain expression of the AP organizer gene Fgf8. Thus, the precise spatial and temporal regulation of Gli2A and Gli3R by Shh is instrumental in coordinating mid/hindbrain development in three dimensions.
Gli3 Coordinates Three-dimensional Patterning and Growth of the Tectum and Cerebellum by Integrating Shh and Fgf8 Signaling
The coordination of anterior-posterior (AP) and dorsal-ventral (DV) patterning of the mesencephalon (mes) and rhombomere 1 (r1) is instrumental for the development of three distinct brain structures: the tectum and cerebellum dorsally and the tegmentum ventrally. Patterning of the mes/r1 is primarily mediated by signaling molecules secreted from two organizers: sonic hedgehog (Shh) from the floor plate (DV) and Fgf8 from the isthmus (AP). Gli3, a zinc-finger transcription factor in the Shh signaling pathway, has been implicated in regulating Fgf8 expression and is therefore a potential candidate for coordinating the action of the two organizers. By inactivating mouse Gli3 at successive embryonic time points in vivo, we uncovered the extent and the underlying mechanism of Gli3 function in the mes/r1. We demonstrate that before E9.0, Gli3 is required for establishing a distinct posterior tectum, isthmus and cerebellum, but does not play a role in the development of the tegmentum. Between E9.0 and E11.0, Gli3 continues to be required for isthmus and cerebellum development, but primarily for defining the cerebellar foliation pattern. We show that Gli3 regulates patterning of the isthmus and cerebellar anlage by confining Fgf8 expression to the isthmus, and attenuates growth of dorsal r1 (before E11.0) and the dorsal mes and isthmus (beyond E11.0) through regulation of cell proliferation and viability. In conclusion, our results show that Gli3 is essential for the coordinated three-dimensional patterning and growth of the dorsal mes/r1.
Sonic Hedgehog is a Chemoattractant for Midbrain Dopaminergic Axons
Midbrain dopaminergic axons project from the substantia nigra (SN) and the ventral tegmental area (VTA) to rostral target tissues, including the striatum, pallidum, and hypothalamus. The axons from the medially located VTA project primarily to more medial target tissues in the forebrain, whereas the more lateral SN axons project to lateral targets including the dorsolateral striatum. This structural diversity underlies the distinct functions of these pathways. Although a number of guidance cues have been implicated in the formation of the distinct axonal projections of the SN and VTA, the molecular basis of their diversity remains unclear. Here we investigate the molecular basis of structural diversity in mDN axonal projections. We find that Sonic Hedgehog (Shh) is expressed at a choice point in the course of the rostral dopaminergic projections. Furthermore, in midbrain explants, dopaminergic projections are attracted to a Shh source. Finally, in mice in which Shh signaling is inactivated during late neuronal development, the most medial dopaminergic projections are deficient. In addition to the role of Shh in the induction of mDN precursors, Shh plays an important role in dopaminergic axon pathfinding to rostral target tissues. Furthermore, Shh signaling is involved in determining the structural diversity of these dopaminergic projections.
Temporal-spatial Changes in Sonic Hedgehog Expression and Signaling Reveal Different Potentials of Ventral Mesencephalic Progenitors to Populate Distinct Ventral Midbrain Nuclei
The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points.
Sonic Hedgehog Lineage in the Mouse Hypothalamus: From Progenitor Domains to Hypothalamic Regions
ABSTRACT: BACKGROUND: The hypothalamus is a brain region with essential functions for homeostasis and energy metabolism, and alterations of its development can contribute to pathological conditions in the adult, like hypertension, diabetes or obesity. However, due to the anatomical complexity of the hypothalamus, its development is not well understood. Sonic hedgehog (Shh) is a key developmental regulator gene expressed in a dynamic pattern in hypothalamic progenitor cells. To obtain insight into hypothalamic organization, we used genetic inducible fate mapping (GIFM) to map the lineages derived from Shh-expressing progenitor domains onto the four rostrocaudally arranged hypothalamic regions: preoptic, anterior, tuberal and mammillary. RESULTS: Shh-expressing progenitors labeled at an early stage (before E9.5) contribute neurons and astrocytes to a large caudal area including the mammillary and posterior tuberal regions as well as tanycytes (specialized median eminence glia). Progenitors labeled at later stages (after E9.5) give rise to neurons and astrocytes of the entire tuberal region and in particular the ventromedial nucleus, but not to cells in the mammillary region and median eminence. At this stage, an additional Shh-expressing domain appears in the preoptic area and contributes mostly astrocytes to the hypothalamus. Shh-expressing progenitors do not contribute to the anterior region at any stage. Finally, we show a gradual shift from neurogenesis to gliogenesis, so that progenitors expressing Shh after E12.5 generate almost exclusively hypothalamic astrocytes. CONCLUSIONS: We define a fate map of the hypothalamus, based on the dynamic expression of Shh in the hypothalamic progenitor zones. We provide evidence that the large neurogenic Shh-expressing progenitor domains of the ventral diencephalon are continuous with those of the midbrain. We demonstrate that the four classical transverse zones of the hypothalamus have clearly defined progenitor domains and that there is little or no cell mixing between the tuberal and anterior or the preoptic and anterior hypothalamus. Finally, we show that, in the tuberal hypothalamus, neurons destined for every mediolateral level are produced during a period of days, in conflict with the current "three-wave" model of hypothalamic neurogenesis. Our work sets the stage for a deeper developmental analysis of this complex and important brain region.
