XB-ART-50080Genesis February 1, 2015; 53 (2): 203-24.
Early development of the neural plate: new roles for apoptosis and for one of its main effectors caspase-3.
Despite its tremendous complexity, the vertebrate nervous system emerges from a homogenous layer of neuroepithelial cells, the neural plate. Its formation relies on the time- and space-controlled progression of developmental programs. Apoptosis is a biological process that removes superfluous and potentially dangerous cells and is implemented through the activation of a molecular pathway conserved during evolution. Apoptosis and an unconventional function of one of its main effectors, caspase-3, contribute to the patterning and growth of the neuroepithelium. Little is known about the intrinsic and extrinsic cues controlling activities of the apoptotic machinery during development. The BarH-like (Barhl) proteins are homeodomain-containing transcription factors. The observations in Caenorhabditis elegans, Xenopus, and mice document that Barhl proteins act in cell survival and as cell type-specific regulators of a caspase-3 function that limits neural progenitor proliferation. In this review, we discuss the roles and regulatory modes of the apoptotic machinery in the development of the neural plate. We focus on the Barhl2, the Sonic Hedgehog, and the Wnt pathways and their activities in neural progenitor survival and proliferation.
PubMed ID: 25619400
Article link: Genesis
Genes referenced: apaf1 axin2 bak1 barhl2 bax bcl2 bcl2l1 bid casp3.2 casp7 casp8 casp9 ccnb1.2 cdc25b cdh1 cdk1 cdkn1a cdkn1b ces3.7 chrd.1 csnk1a1 csnk1g2 ctnnb1 diablo dvl2 fadd gsk3b kif11 krt8.1 lrp5 mcl1 myc mycn nsg1 ptch1 shh smo sox3 tbx2 tgfb1 tp53 tradd wnt3 wnt3a
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|FIG. 1. Scheme of the vertebrate apoptotic pathway. In a viable cell, the proapoptotic proteins Bax and Bak of the B-cell lymphoma 2 (Bcl2) family are repressed by the antiapoptotic proteins Bcl2, Mcl1 and BclXL. Proapoptotic stimuli such as cytokine deprivation or endoplasmic reticulum stress trigger the induction of the BH3-only proteins. BH3-only proteins counteract the effect of Bcl2 and Bcl-XL, a process that leads to the release of Bax and Bak. Bax and Bak are then inserted and activated at the outer mitochondrial membrane where they promote the formation of pores. A key event in the induction of death is the release of cytochrome c and SMAC/Diablo from the mitochondrial intermembranous space to the cytoplasm. SMAC/Diablo inhibits the IAP proteins, whereas cytochrome c recruits APAF-1 to form the apoptosome. Caspase-9 is activated by the apoptosome that leads to the cleavage of the effector caspases, caspase-3 and caspase- 7, and to irreversible death. In parallel to this intrinsic pathway, the stimulation of death receptors leads to the recruitment of the adaptor proteins such as TRADD and FADD through their death domain. Caspase-8 is then recruited to the membrane dimerizes and can directly activate caspase-3 and caspase-7. Crosstalk between the extrinsic and the intrinsic pathway depends on the caspase-8 cleavage of the BH3-only protein BH3-interacting domain death agonist BID. The product of this cleavage t-BID (truncated BID) inhibits Bcl2, Mcl1 and Bcl-XL and promotes the accumulation of Bax and Bak. Both pathways can be activated through p53 stimulation resulting from DNA damage. Another pathway is linked to the absence of the survival factor Shh that leads to the stimulation of the proapoptotic function of its receptor patched. In the absence of its ligand, patched forms a protein complex with the adaptor protein DRAL (downregulated in rhabomuosarcoma LIM-domain protein) and one caspase recruitment (CARD)-domain containing protein, either TUCAN (family member, 8) or NALP (NLR family, pyrin domain containing 1). Apical caspase-9 is then recruited to this complex and activated by patched, which leads to activation of the effector caspases.|
|FIG. 2. Barhl2: a regulator of EPCD in the neural plate. (a) Expression patterns of barhl2, shh, and chordin and pattern of endogenous apoptosis in Xenopus neurula embryos. The schematic representation of barhl2, shh, and endogenous apoptosis in the developing neural plate: At stage 14 barhl2 is expressed in the caudal forebrain primordium as two bilateral strips and in cells bordering the floor plate and possibly the notochord (yellow in the scheme). The axial organizer, which we define as the notochord, the prechordal plate and the floor plate, emerge from the Spemann organizer and secrete anti-BMP factors (chordin) and Shh (lavender). The secretion is shown with arrows and cells undergoing apoptosis are shown as black spots. Whole mount in situ hybridization (ISH) using barhl2, shh, or chordin as probes and whole mount TUNEL staining as indicated. Representative neurula embryos are shown in dorsal view, anterior up. Cells undergoing endogenous apoptosis are marked in blue. The schematic representation of the role of Barhl2 on the axial organizer size: In wild-type embryos, Barhl2 has a proapoptotic role (black cells) and negatively regulates the number of axial organizer cells that secrete anti-BMP factors and Shh (pink). As a result, Shh and BMP activity is reduced within the neural plate. When Barhl2 activity is depleted, more cells secreting Shh and chordin survive. This leads to enhanced Shh (blue) and BMP (orange) signaling activities and subsequently to an increase in the neural plate size marked by Sox3 (green), (after Offner et al., 2005). (b) The BarH survival pathways in amphibians and nematodes. During neurulation, Barhl2 regulates EPCD. Barhl2 interacts with Groucho and induces cell death of axial organizer cells expressing the morphogens Shh and chordin through an unknown mechanism. In C. elegans, the Barhl2 orthologue, Ceh-30, regulates the survival of CEM chemosensory neurons in male organisms. In males, ceh-30 is induced by cell-specific signals, and it interacts with UNC-37, the groucho orthologue, and acts as a transcriptional repressor of ced-3 and egl-1 genes.|
|FIG. 3. Scheme illustrating cell-cycle dynamics in neural progenitors. The neuroepithelium is composed of a single layer of rapidly dividing NSCs and progenitors. As the cells adjacent to the lumen continue to proliferate, the neural progenitors migrate and form a second layer around the neural tube. This layer becomes progressively thicker and is called the mantle zone (MZ), whereas the germinal neuroepithelium is known as the ventricular zone (VZ), subsequently called the ependyma. Cells in the MZ differentiate into both neurons and glia. In the germinal neuroepithelium, neural progenitors undergo different phases of the cell cycle. The nucleus of proliferating cells undergoes a process of interkinetic nuclear migration that corresponds to each phase of the cycle. The nuclei move away from the apical surface during G1. DNA replication (S phase) takes place in the basal VZ. During G2, the nuclei migrate away from the apical ventricular zone where they undergo mitosis (M). After one cycle is completed, the cells choose either to re-enter the cell cycle or to exit and differentiate. Quiescent cells are found in the MZ. Progression through the cell cycle depends on the cyclin/CDK complexes. During G1, phosphorylation of the retinoblastoma complex (Rb) by cyclins D releases the transcription factor E2F and induces the expression of cyclins E. DNA synthesis during the S phase is followed by the verification of DNA integrity. The progression through mitosis depends on the cyclin B/CDK1 complex. The CDK inhibitors (CKI) p27/Kip1 and p21/Cip1 stop the progression through the cycle and favor exit from the cell cycle (G0).|
|FIG. 4. The Wnt canonical and the Shh pathways and their interactions. (a) Schematic representation of the Shh pathway. In the absence of Shh, patched (Ptc) inhibits smoothened (Smo) and prevents its translocation to the cell surface. Cos2 binding to the microtubules allows the formation of a protein complex with recruitment of GSK3, CK1a, PKA, and Su(Fu). The protein complex phosphorylates the Gli proteins and targets their proteosomal degradation. Truncated Gli forms translocate to the nucleus where they repress target gene expression. In parallel, patched can recruit and activate caspase-9 a process that leads to cell death. Binding of Shh to Ptc initiates the pathway, alleviates repression of Smo, which is translocated to the membrane, and promotes cell survival. Cos2 binds to Smo and inhibits the phosphorylation of Gli proteins by releasing the different kinases. Full activation of the pathway is achieved by phosphorylation of the cytosolic C-tail of Smo by PKA and CK1. Full-length forms of Gli are translocated to the nucleus where they induce the expression of Shh target genes such as Gli- 1, Ptc, N-Myc, Bcl-2, and cyclin D1. (b) The schematic representation of the Wnt canonical pathway. In the absence of a Wnt signal, bcatenin is phosphorylated and targeted for proteasome-mediated degradation by a protein complex containing GSK3-b, CK1a, APC, and Axin. After binding of the Wnt ligand to the receptors Frizzled (Fz) and LRP5/6, dishevelled (Dvl) binds to Fz and recruits the destruction complex at the cytosolic tail of LRP5/6. LRP5/6 tail is then phosphorylated by GSK3-b and bound by Axin. In this context, b-catenin is stabilized and translocated to the nucleus where in association with TCF/LEF transcription factors it stimulates expression of the target genes c-myc, axin2, and cyclin D1. In parallel, b-catenin is present at the adherens junctions where it associates with cadherins and with the cytoskeleton, allowing adhesion between neighboring cells. Caspases, and specifically caspase-3, can destabilize the adherens junctions in part by inhibiting b-catenin association with E-cadherin. At note in vitro b-catenin is a substrate for caspases. (c) Crosstalk between the Wnt and the Shh pathways promotes cell-cycle progression. The Shh pathway mostly acts on the G2 and S phases through the regulation of phosphatase CDC25B expression. N-Myc is capable of regulating cyclin D1 expression, which allows progression through G1. In some cases, Shh also regulates the expression of late cyclins A and B. The Wnt canonical pathway also promotes G1 progression by inducing the expression of c-myc, which regulates cyclin D1 expression and inhibits p27/Kip1 and p21/Cip1. A feedback loop is regulated by cyclins Y, which phosphorylate the cytosolic tail of LRP5/6 to activate the Wnt pathway.|
|FIG. 5. Barhl2 acts as a brake on diencephalic progenitor proliferation. (a) barhl2, wnt, and shh ligands are coexpressed in the diencephalic primordium. The scheme of a stage 27 neural tube: barhl2 (yellow) is expressed in the prosomere p2 (p2) within the diencephalon, in a territory giving rise to the ZLI, a secondary organizer that secretes Shh (blue). Shh is also expressed in the anterior neural tube basal plate. Wnt3a (in red) as Wnt3 (data not shown) is expressed in the dorsal (alar plate) part of the prosomere p2, in the midbrain roof plate. The two other major secondary organizers, the ANR (pink) and the isthmic organizer (isO, green), that play roles in patterning the forebrain and the midbrain/hindbrain, respectively, are also shown. The secretion is represented with arrows. Whole mount ISH using barhl2, shh, or wnt3a probes as indicated. Representative dissected neural tube of wild-type embryos is shown, dorsal view anterior left. Whole mount TUNEL analysis: cells undergoing endogenous apoptosis appear in blue. (b) Nonapoptotic functions of caspases regulate the levels and activity of b-catenin. In Xenopus diencephalic primordium, Barhl2 acts upstream of a nonapoptotic function of caspase-3 that limits both levels and activity of b-catenin and consequently limits the proliferation of neural progenitors, controls the neuroepithelium architecture, and regulates the transcription of Wnt target genes. In drosophila SOP cells, the caspases orthologues DRONC and drICE limit the activity of the Wingless pathway by cleaving an isoform of GSK3-b, Shaggy, which destabilizes Armadillo (b-catenin orthologue) and consequently limits the Wnt pathway.|