Calcium Channel Subunit α2δ4 Is Regulated by Early Growth Response 1 and Facilitates Epileptogenesis.

Transient brain insults, including status epilepticus (SE), can trigger a period of epileptogenesis during which functional and structural reorganization of neuronal networks occurs resulting in the onset of focal epileptic seizures. In recent years, mechanisms that regulate the dynamic transcription of individual genes during epileptogenesis and thereby contribute to the development of a hyperexcitable neuronal network have been elucidated. Our own results have shown early growth response 1 (Egr1) to transiently increase expression of the T-type voltage-dependent Ca2+ channel (VDCC) subunit CaV3.2, a key proepileptogenic protein. However, epileptogenesis involves complex and dynamic transcriptomic alterations; and so far, our understanding of the transcriptional control mechanism of gene regulatory networks that act in the same processes is limited. Here, we have analyzed whether Egr1 acts as a key transcriptional regulator for genes contributing to the development of hyperexcitability during epileptogenesis. We found Egr1 to drive the expression of the VDCC subunit α2δ4, which was augmented early and persistently after pilocarpine-induced SE. Furthermore, we show that increasing levels of α2δ4 in the CA1 region of the hippocampus elevate seizure susceptibility of mice by slightly decreasing local network activity. Interestingly, we also detected increased expression levels of Egr1 and α2δ4 in human hippocampal biopsies obtained from epilepsy surgery. In conclusion, Egr1 controls the abundance of the VDCC subunits CaV3.2 and α2δ4, which act synergistically in epileptogenesis, and thereby contributes to a seizure-induced "transcriptional Ca2+ channelopathy."SIGNIFICANCE STATEMENT The onset of focal recurrent seizures often occurs after an epileptogenic process induced by transient insults to the brain. Recently, transcriptional control mechanisms for individual genes involved in converting neurons hyperexcitable have been identified, including early growth response 1 (Egr1), which activates transcription of the T-type Ca2+ channel subunit CaV3.2. Here, we find Egr1 to regulate also the expression of the voltage-dependent Ca2+ channel subunit α2δ4, which was augmented after pilocarpine- and kainic acid-induced status epilepticus. In addition, we observed that α2δ4 affected spontaneous network activity and the susceptibility for seizure induction. Furthermore, we detected corresponding dynamics in human biopsies from epilepsy patients. In conclusion, Egr1 orchestrates a seizure-induced "transcriptional Ca2+ channelopathy" consisting of CaV3.2 and α2δ4, which act synergistically in epileptogenesis.

The zinc-finger transcription factor GLI3 is a regulator of precerebellar neuronal migration.

Hindbrain precerebellar neurons arise from progenitor pools at the dorsal edge of the embryonic hindbrain: the caudal rhombic lip. These neurons follow distinct migratory routes to establish nuclei that provide climbing or mossy fiber inputs to the cerebellum. Gli3, a zinc-finger transcription factor in the Sonic hedgehog signaling pathway, is an important regulator of dorsal brain development. We demonstrate that in Gli3-null mutant mice, disrupted neuronal migratory streams lead to a disorganization of precerebellar nuclei. Precerebellar progenitors are properly established in Gli3-null embryos and, using conditional gene inactivation, we provide evidence that Gli3 does not play a cell-autonomous role in migrating precerebellar neurons. Thus, GLI3 likely regulates the development of other hindbrain structures, such as non-precerebellar nuclei or cranial ganglia and their respective projections, which may in turn influence precerebellar migration. Although the organization of non-precerebellar hindbrain nuclei appears to be largely unaffected in absence of Gli3, trigeminal ganglia and their central descending tracts are disrupted. We show that rostrally migrating precerebellar neurons are normally in close contact with these tracts, but are detached in Gli3-null embryos.

In vitro segregation and isolation of human pluripotent stem cell-derived neural crest cells.

The neural crest (NC) is a transient embryonic cell population with remarkable characteristics. After delaminating from the neural tube, NC cells (NCCs) migrate extensively, populate nearly every tissue of the body and differentiate into highly diverse cell types such as peripheral neurons and glia, but also mesenchymal cells including chondrocytes, osteocytes, and adipocytes. While the NC has been extensively studied in several animal models, little is known about human NC development. A number of methods have been established to derive NCCs in vitro from human pluripotent stem cells (hPSC). Typically, these protocols comprise several cell culture steps to enrich for NCCs in the neural derivatives of the differentiating hPSCs. Here we report on a remarkable and hitherto unnoticed in vitro segregation phenomenon that enables direct extraction of virtually pure NCCs during the earliest stages of hPSC differentiation. Upon aggregation to embryoid bodies (EB) and replating, differentiating hPSCs give rise to a population of NCCs, which spontaneously segregate from the EB outgrowth to form conspicuous, macroscopically visible atoll-shaped clusters in the periphery of the EB outgrowth. Isolation of these NC clusters yields p75NTR(+)/SOXE(+) NCCs, which differentiate to peripheral neurons and glia as well as mesenchymal derivatives. Our data indicate that differentiating hPSC cultures recapitulate, in a simplified manner, the physical segregation of central nervous system (CNS) tissue and NCCs. This phenomenon may be exploited for NCC purification and for studying segregation and differentiation processes observed during early human NC development in vitro.

Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain.

Midbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia.

Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei.

BACKGROUND: 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. RESULTS: We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes. CONCLUSIONS: We define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.

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.

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.

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.

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.

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.

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.