Expression of the Hippo pathway effector, TEAD1, within the developing murine forebrain

Mind Bomb 1-Expressing Intermediate Progenitors Generate Notch Signaling to Maintain Radial Glial Cells

Decision letter: Exploring the role of the outer subventricular zone during cortical folding through a physics-based model

Editor's evaluation: Exploring the role of the outer subventricular zone during cortical folding through a physics-based model

The Hippo pathway is conserved through evolution and plays critical roles in development, tissue homeostasis and tumorigenesis. Yes-associated protein (YAP) is a transcriptional coactivator downstream of the Hippo pathway. Previous studies have demonstrated that activation of YAP promotes proliferation in the developing brain. Whether YAP is required for the production of neural progenitor cells or neurons in vivo remains unclear. We demonstrated that SATB homeobox 2 (SATB2)-positive projection neurons (PNs) in upper layers, but not T-box brain transcription factor 1-positive and Coup-TF interacting protein 2-positive PNs in deep layers, were decreased in the neonatal cerebral cortex of Yap conditional knockout (cKO) mice driven by Nestin-Cre. Cell proliferation was reduced in the developing cerebral cortex of Yap-cKO. SATB2-positive PNs are largely generated from intermediate progenitor cells (IPCs), which are derived from radial glial cells (RGCs) during cortical development. Among these progenitor cells, IPCs but not RGCs were decreased in Yap-cKO. We further demonstrated that cell cycle re-entry was reduced in progenitor cells of Yap-cKO, suggesting that fewer IPCs were generated in Yap-cKO. YAP is required for the production of IPCs and upper-layer SATB2-positive PNs during development of the cerebral cortex in mice.

Two distinct populations of cerebral cortical progenitor cells that generate neurons during embryogenesis have been identified: radial glial cells and intermediate progenitor cells. Despite advances in our understanding of progenitor cell populations, we know relatively little about factors that regulate their proliferative behaviour. 17-beta-Estradiol (E2) is present in the adult and developing mammalian brain, and plays an important role in central nervous system processes such as neuronal differentiation, survival and plasticity. E2 also stimulates neurogenesis in the adult dentate gyrus. We examined the role of E2 during embryonic cortical neurogenesis through immunohistochemistry, in situ hybridization, functional enzyme assay, organotypic culture and in utero administration of estradiol-blocking agents in mice. We show that aromatase, the E2 synthesizing enzyme, is present in the embryonic neocortex, that estrogen receptor-alpha is present in progenitor cells during cortical neurogenesis, that in vitro E2 administration rapidly promotes proliferation, and that in utero blockade of estrogen receptors decreases proliferation of embryonic cortical progenitor cells. Furthermore, the E2 inhibitor alpha-fetoprotein is expressed at high levels by radial glial cells but at lower levels by intermediate progenitor cells, suggesting that E2 differentially influences the proliferation of these cortical progenitor cell types. These findings demonstrate a new functional role for E2 as a proliferative agent during critical stages of cerebral cortex development.

Yap1 Acts Downstream of α-Catenin to Control Epidermal Proliferation

The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 --> Tbr2 --> Tbr1 in the differentiation of radial glia --> intermediate progenitor cell --> postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia --> postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.

The hippo signaling pathway is a highly conserved evolutionary signaling pathway that plays an important role in regulating cell proliferation, organ size, tissue development, and regeneration. Increasing evidences consider that the hippo signaling pathway is involved in the process of respiratory diseases. Hippo signaling pathway is mainly composed of mammalian STE20-like kinase 1/2 (MST1/2), large tumor suppressor 1/2 (LATS1/2), WW domain of the Sav family containing protein 1 (SAV1), MOB kinase activator 1 (MOB1), Yes-associated protein (YAP) or transcriptional coactivator with PDZ-binding motif (TAZ), and members of the TEA domain (TEAD) family. YAP is the cascade effector of the hippo signaling pathway. The activation of YAP promotes pulmonary arterial vascular smooth muscle cells (PAVSMCs) proliferation, which leads to pulmonary vascular remodeling; thereby the pulmonary arterial hypertension (PAH) is aggravated. While the loss of YAP leads to high expression of inflammatory genes and the accumulation of inflammatory cells, the pneumonia is consequently exacerbated. In addition, overexpressed YAP promotes the proliferation of lung fibroblasts and collagen deposition; thereby the idiopathic pulmonary fibrosis (IPF) is promoted. Moreover, YAP knockout reduces collagen deposition and the senescence of adult alveolar epithelial cells (AECs); hence the IPF is slowed. In addition, hippo signaling pathway may be involved in the repair of acute lung injury (ALI) by promoting the proliferation and differentiation of lung epithelial progenitor cells and intervening in the repair of pulmonary capillary endothelium. Moreover, the hippo signaling pathway is involved in asthma. In conclusion, the hippo signaling pathway is involved in respiratory diseases. More researches are needed to focus on the molecular mechanisms by which the hippo signaling pathway participates in respiratory diseases.

The Hippo pathway is a critical regulator of tissue size, and aberrations in pathway regulation lead to cancer. MST1/2 and LATS1/2 kinases comprise the core of the pathway that, in association with adaptor proteins SAV and MOB, functions in a sequential manner to phosphorylate and inhibit the transcription factors YAP and TAZ. Here we identify mammalian MARK family members as activators of YAP/TAZ. We show that depletion of MARK4 in MDA-MB-231 breast cancer cells results in the loss of nuclear YAP/TAZ and decreases the expression of YAP/TAZ targets. We demonstrate that MARK4 can bind to MST and SAV, leading to their phosphorylation, and that MARK4 expression attenuates the formation of a complex between MST/SAV and LATS, which depends on the kinase activity of MARK4. Abrogation of MARK4 expression using siRNAs and CRISPR/Cas9 gene editing attenuates the proliferation and migration of MDA-MB-231 cells. Our results show that MARK4 acts as a negative regulator of the Hippo kinase cassette to promote YAP/TAZ activity and that loss of MARK4 restrains the tumorigenic properties of breast cancer cells.

Mammalian corticogenesis occurs through a complex process that includes neurogenesis, in which neural progenitor cells proliferate, differentiate, and migrate. It has been reported recently that neurogenesis occurs in the subventricular zone (SVZ), a region previously thought to be the primary site of gliogenesis. It has been recognized that in the SVZ, intermediate progenitor cells, derived from radial glial cells that are multipotent neural stem cells, produce only neurons. However, the molecular mechanisms underlying the regulation of neural stem cells and intermediate progenitor cells as well as their contribution to overall corticogenesis remain unknown. The docking protein FRS2alpha is a major mediator of signaling by means of FGFs and neurotrophins. FRS2alpha mediates many of its pleiotropic cellular responses by recruiting the adaptor protein Grb2 and the protein tyrosine phosphatase Shp2 upon ligand stimulation. Here, we report that targeted disruption of Shp2-binding sites in FRS2alpha leads to severe impairment in cerebral cortex development in mutant mice. The defect in corticogenesis appears to be due at least in part to abnormalities in intermediate progenitor cells. Genetic evidence is provided that FRS2alpha plays critical roles in the maintenance of intermediate progenitor cells and in neurogenesis in the cerebral cortex. Moreover, FGF2-responsive neurospheres, which are cell aggregates derived from neural stem/progenitor cells (NSPCs), from FRS2alpha mutant mice were smaller than those of WT mice. However, mutant NSPCs were able to self-renew, demonstrating that Shp2-binding sites on FRS2alpha play an important role in NSPC proliferation but are dispensable for NSPC self-renewing capacity after FGF2 stimulation.

The Hippo signalling cascade is an evolutionarily conserved pathway critical for the development of numerous organ systems and is required for the development of many parts of the mammalian nervous system, including the cerebellum. The Hippo pathway converges, via the nuclear YAP/TAZ co-transcription factors, on transcription factors of the TEA Domain (TEAD) family (TEAD1-4) and promotes the expression of pro-proliferative genes. Despite the importance of TEAD function, our understanding of spatial and temporal expression of this family is limited, as is our understanding of which TEAD family members regulate Hippo-dependent organ development. Here, we focus on TEAD1 and how this factor contributes to postnatal murine cerebellar development. We find expression of TEAD1 within cerebellar progenitor cells and glial cells, including astrocytes and Bergmann glia, as well as by some interneurons within the granular layer. The importance of TEAD1 expression for cerebellar development was investigated using a conditional ablation approach, which revealed a range of developmental deficits in Tead1 mutants, including an underdeveloped cerebellum, morphological defects in Bergmann Glia and Purkinje Neurons, as well as granule neuron migration defects. Collectively, these findings suggest a major role for TEAD1 as an effector of the Hippo pathway during cerebellar development.

Recent work has begun to identify neural stem and progenitor cells in the embryonic and adult brain, and is unravelling the mechanisms whereby new nerve cells are created and delivered to their correct locations. Radial glial (RG) cells, which are present in the developing mammalian brain, have been proposed to be neural stem cells because they produce multiple cell types. Furthermore, time-lapse imaging demonstrates that RG cells undergo asymmetric self-renewing divisions to produce immature neurons that migrate along their parent radial fibre to reach the developing cerebral cortex. RG cells also produce intermediate progenitor (IP) cells that undergo symmetric division in the subventricular zone of the embryonic cortex to produce pairs of neurons. The symmetric IP divisions increase cell number within the same cortical layer. This two-step process of neurogenesis suggests new mechanisms for the generation of cell diversity and cell number in the developing cortex and supports a model similar to that proposed for the development of the fruit fly CNS. In this model, a temporal sequence of gene expression changes in asymmetrically dividing self-renewed RG cells could lead to the differential inheritance of cell identity genes in cortical cells generated at different cell cycles.

RP58 Regulates the Multipolar-Bipolar Transition of Newborn Neurons in the Developing Cerebral Cortex

7,12-Dimethylbenz(a)anthracene (DMBA) is a polycyclic aromatic hydrocarbon formed by the combustion of organic substances and has ovotoxic and carcinogenic effects on ovarian follicular development in rodents. The Hippo signaling pathway contributes to the understanding of various molecular mechanisms, including cell proliferation, differentiation, apoptosis, organ size regulation, and tumorigenesis, as an evolutionarily conserved pathway. In this study, we hypothesized that the Hippo signaling pathway may play a role in the mechanism of DMBA-induced ovotoxicity. We aimed to identify Hippo signaling pathway proteins in DMBA-treated ovaries via immunohistochemistry and quantitative real-time PCR (qRT-PCR). Twenty-eight-day-old 18 BalbC female mice were used and divided into three groups. The control group received no treatment, the vehicle group was injected daily with sesame oil, and the DMBA group was injected intraperitoneally with 1 mg/kg/day DMBA (dissolved in sesame oil) for 14 consecutive days. The sections were subjected to periodic acid-Schiff (PAS) staining to demonstrate the morphological differences between the DMBA and control groups of mouse ovaries. Anti-Mullerian hormone (AMH) serum levels were analyzed via ELISA, and follicles were counted to evaluate the follicle reserve. Immunohistochemistry was performed to determine the localization of the Hippo signaling pathway proteins MST1/2, LATS1/2, YAP1, and TEAD4 and to assess oxidative stress with a nitrotyrosine (NTY) antibody. The mRNA levels of Hippo signaling components were detected via qRT-PCR. The Hippo signaling pathway may play a role in the mechanisms underlying the rapid depletion of follicles through increased oxidative stress, an increased number of atretic follicles, and a decreased number of corpus luteum in DMBA-induced ovotoxicity. We demonstrated that in the DMBA-induced ovotoxicity model, the Hippo signaling pathway is inactivated by YAP/TAZ translocation to the nucleus, and the increase in YAP1 and TEAD4 expression is at the translational level. This study provides the first evidence of the relationship between ovotoxicity and the Hippo signaling pathway in DMBA-induced ovotoxicity in mice. Therefore, our findings suggest that the Hippo pathway could be pharmacologically targeted to regulate DMBA-induced ovotoxicity.

Event Abstract Back to Event The origin and migration of cortical neurons in human and mouse Laura A. Elias1*, David V. Hansen1, Arnold R. Kriegstein1 and Jan H. Lui1 1 University of California, Department of Signal Processing, United States The adult cerebral cortex is composed of excitatory and inhibitory neurons that arise from progenitor cells in disparate proliferative regions in the developing brain and follow different migratory paths. Excitatory pyramidal neurons originate near the ventricle and migrate radially to their positions in the cortical plate. Inhibitory interneurons arise in the ventral telencephalon and migrate tangentially to enter the developing cortex before migrating radially to reach their correct laminar position. Radial glia produce both excitatory and inhibitory neurons indirectly, by generating intermediate progenitor cells that undergo symmetric cell divisions to increase the numbers of nerve cells they produce. In the rodent, intermediate progenitor cells generally reside in a subventricular zone (SVZ). We found that in the embryonic human cortex, very large numbers of neuronal stem and intermediate progenitor cells are produced in a proliferative zone not present in the rodent brain, the outer subventricular zone. The medial ganglionic eminence in the fetal human brain also contains a large SVZ and is the source of large numbers of inhibitory cortical neurons. The oSVZ may account for the very large neocortex size peculiar to humans. Gap junction adhesion has recently been shown to play an important mechanistic role in the radial migration of excitatory neurons. Gap junctions are expressed in stem and progenitor cells of the ventral telencephalon as well as migrating neurons. By allowing communication among progenitors or between progenitors and neuroblasts, gap junctions are thought to play a role in regulating cortical development. We asked whether a gap junction-mediated mechanism governs the tangential or radial migration of inhibitory interneurons. Using shRNA knockdown of Cx43 and Cx26 together with rescue experiments, we found that gap junctions are dispensible for the tangential migration of interneurons, but that Cx43 plays a role in the switch from tangential to radial migration that allows interneurons to enter the cortical plate and find their correct laminar position. Moreover, this action is dependent on the adhesive properties and the C-terminus of Cx43, but not the Cx43 channel. Thus, the radial phase of interneuron migration resembles that of excitatory neuron migration in terms of dependence on Cx43 adhesion. These findings also provide mechanistic support for an interaction between migrating interneurons and radial glia during the switch from tangential to radial migration.