1)What is the evidence that the expression of pax6 confers competence to form ey
ID: 33910 • Letter: 1
Question
1)What is the evidence that the expression of pax6 confers competence to form eyes.
2)In the WNT signaling pathway GSK-3 normally inhibits -catenin, keeping it in the cytoplasm so that it cannot go into the nucleus to activate transcription. (6pt)
a) A null mutation in GSK-3 would have what affect on the WNT signaling pathway?
b) When DSH is activated, it inhibits GSK-3. What affect would a null mutation in DSH have on the pathway?
c) If you have null mutations in both DSH and GSK-3, what would be the affect on the pathway?
3)What are four affects that signaling from a ligand will have on a cell (what will the ligand cause the cell to do?)?
4)Explain how lateral inhibition works.
5)Describe how the anterior localization of bicoid mRNA and the posterior localization of the nanos mRNA determine eventual banding pattern of the even-skipped gene.
6)Why do mutations in some segment polarity genes result in segments that are mirror images of other segments?
7)What did mosaic analysis tell us about the role of sevenless in the R7 cell and boss in the R8 cell?
8)Describe the role of Maternal Affect Genes, Gap Genes, and Pair-rule genes in the anterior-posterior patterning of the Drosophila embryo.
9)What is a morphogen? How do morphogens affect patterning of Drosophila segments?
10)What are the common features of signaling pathways?
Explanation / Answer
1) After lentectomy, larval Xenopus laevis can regenerate a new lens by transdifferentiation of the outer cornea and pericorneal epidermis (lentogenic area). This process is promoted by retinal factor(s) accumulated into the vitreous chamber. To understand the molecular basis of the lens-regenerating competence (i.e. the capacity to respond to the retinal factor forming a new lens) in the outer cornea and epidermis, we analysed the expression of otx2, pax6,sox3, pitx3, prox1, ?B1-cry (genes all involved in lens development) by Real-time RT-PCR in the cornea and epidermis fragments dissected from donor larvae. The same fragments were also implanted into the vitreous chamber of host larvae to ascertain their lens-regenerating competence using specific anti-lens antibodies. The results demonstrate that there is a tight correlation between lens-regenerating competence and pax6 expression. In fact, (1) pax6 is the only one of the aforesaid genes to be expressed in the lentogenic area; (2) pax6 expression is absent in head epidermis outside the lentogenic area and in flank epidermis, both incapable of transdifferentiating into lens after implantation into the vitreous chamber; (3) in larvae that have undergone eye transplantation under the head or flank epidermis, pax6 re-expression was observed only in the head epidermis covering the transplanted eye. This is consistent with the fact that only the head epidermis reacquires the lens-regenerating competence after eye transplantation, forming a lens following implantation into the vitreous chamber; and (4) in larvae that have undergone removal of the eye, the epidermis covering the orbit maintained pax6 expression. This is consistent with the fact that after the eye enucleation the lentogenic area maintains the lens-regenerating competence, giving rise to a lens after implantation into the vitreous chamber. Moreover, we observed that misexpression of pax6 is sufficient to promote the acquisition of the lens-regenerating competence in flank epidermis. In fact, flank epidermis fragments dissected from pax6 RNA injected embryos could form lenses when implanted into the vitreous chamber. The data indicate for the first time that pax6 is a pivotal factor of lens-regenerating competence in the outer cornea and epidermis of larval X. laevis.
Wnt proteins are secreted morphogens that are required for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. There are at least three different Wnt pathways: the canonical pathway, the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway.
In the canonical Wnt pathway, the major effect of Wnt ligand binding to its receptor is the stabilization of cytoplasmic beta-catenin through inhibition of the bea-catenin degradation complex. Beta-catenin is then free to enter the nucleus and activate Wnt-regulated genes through its interaction with TCF (T-cell factor) family transcription factors and concomitant recruitment of coactivators.
Planar cell polarity (PCP) signaling leads to the activation of the small GTPases RHOA (RAS homologue gene-family member A) and RAC1, which activate the stress kinase JNK (Jun N-terminal kinase) and ROCK (RHO-associated coiled-coil-containing protein kinase 1) and leads to remodelling of the cytoskeleton and changes in cell adhesion and motility.
2) OneofthekinasesthatdirectlyphosphorylateGSK-3is AKT/PKB. Native Akt/PKB is inactive, but the kinase can be rapidly activated by phosphatidylinositol 3-kinases, which leads to phosphorylation of Akt/PKB at threonine-308 and serine-473 (21). For full activity, phosphorylation of both sites (threonine- 308 and serine-473) is required. As reported previously, both sites, threonine-308 and serine-473, are phosphorylated in re- sponse to IR both in the cytoplasm and in the nucleus ) . This phosphorylation of Akt/PKB is independent of ATM because Akt/PKB was also phosphorylated in cells from AT patients . Although the phosphorylation of Akt/ PKBafterIRandthefactthatGSK-3isatargetpointedtoward a contribution of Akt/PKB in the signaling chain leading to GSK-3 phosphorylation, we were puzzled by the fact that Akt/PKB is known as a primarily cytoplasmic kinase. DNA lesions and accumulation of p53 occur, though, in the nuclear compartment of the cell. To resolve this caveat, we prepared cytoplasmicandnuclearlysatesanddeterminedtheamountand phosphorylation of Akt/PKB and GSK-3 in cytoplasmic and nuclear fractions. Notably, a significant part of Akt/PKB was localized in the nucleus, although cytoplasmic Akt/PKB protein levels were considerably higher
Most interestingly, nuclear Akt/PKB was phosphorylated at serine-473 at much earlier time points after IR than the cytoplasmic protein and preceded GSK-3 phosphorylation. Moreover, the overall in- crease in phosphorylation was significantly higher for nuclear Akt/PKB. Correspondingly, phosphorylation of GSK-3 was clearly stronger in the nuclear compartment than in the cyto- plasm. Cytoplasmic Akt/PKB was also phosphorylated at thre- onine-308 in response to IR, although phosphorylation of thre- onine-308wasalwaysweakerthanphosphorylationofserine-473 and usually did not exceed a factor of 2. However, we failed to detectasignificantincreaseinphosphorylationofthreonine-308 in the nucleus. ). This phosphorylation of GSK-3
is independent of ATM because it occurs in wild-type and AT cells to the same extent (Fig. 3C). Inhibition of GSK-3 , e.g., with the specific GSK-3 inhibitor alsterpaullone, increased p53 abundance (Fig. 3A) (17), indicating that GSK-3 activity is required for p53 turnover. In consistency with this principle, overexpression of a constitutivelyactivemutantofGSK-3 whereserine-9hadbeen replaced with an alanine, and which is therefore refractory to radiation-mediatedinhibition,significantlyreducedtheaccumu- lation of p53 in response to IR (Fig. 3D) (17). Moreover, in the presenceofanMdm2mutant,inwhichoneorbothoftheGSK-3
phosphorylationsiteshasbeenreplacedwithanasparticacidand thus resembles phosphorylated Mdm2, p53 accumulation after IR was significantly reduced (Fig. 3E). It should also be men- tionedthattheS254Dmutantwasincapableoftargetingp53for degradation and the S240D/S254D was less competent in reduc- ing p53 levels than wild-type Mdm2, indicating that not only the chargebutalsotheaminoacidbackboneisimportantforMdm2 function. Likewise, when we replaced one of the GSK-3 phos- phorylation sites together with another phosphorylated site of Mdm2(16)orwithoneoftheGSK-3primingsites,with an aspartic acid accumulation of p53 after IR was similarly reduced, indicating that hypophosphorylation of the central domain of Mdm2 is crucial for the accumulation of p53 after IR (16).
Accumulation of p53 in Response to IR Requires Akt2/PKB
and DNA-PK. OneofthekinasesthatdirectlyphosphorylateGSK-3is AKT/PKB. Native Akt/PKB is inactive, but the kinase can be rapidly activated by phosphatidylinositol 3-kinases, which leads to phosphorylation of Akt/PKB at threonine-308 and serine-473 (21). For full activity, phosphorylation of both sites (threonine- 308 and serine-473) is required. As reported previously, both sites, threonine-308 and serine-473, are phosphorylated in re- sponse to IR both in the cytoplasm and in the nucleus (22, 23). This phosphorylation of Akt/PKB is independent of ATM because Akt/PKB was also phosphorylated in cells from AT patients (Fig. 4B). Although the phosphorylation of Akt/ PKBafterIRandthefactthatGSK-3isatargetpointedtoward a contribution of Akt/PKB in the signaling chain leading to GSK-3 phosphorylation, we were puzzled by the fact that Akt/PKB is known as a primarily cytoplasmic kinase. DNA lesions and accumulation of p53 occur, though, in the nuclear compartment of the cell. To resolve this caveat, we prepared cytoplasmicandnuclearlysatesanddeterminedtheamountand phosphorylation of Akt/PKB and GSK-3 in cytoplasmic and nuclear fractions. Notably, a significant part of Akt/PKB was localized in the nucleus, although cytoplasmic Akt/PKB protein levels were considerably higher Most interestingly, nuclear Akt/PKB was phosphorylated at serine-473 at much earlier time points after IR than the cytoplasmic protein and preceded GSK-3 phosphorylation. Moreover, the overall in- crease in phosphorylation was significantly higher for nuclear Akt/PKB. Correspondingly, phosphorylation of GSK-3 was clearly stronger in the nuclear compartment than in the cyto- plasm. Cytoplasmic Akt/PKB was also phosphorylated at thre- onine-308 in response to IR, although phosphorylation of thre- onine-308wasalwaysweakerthanphosphorylationofserine-473 and usually did not exceed a factor of 2. However, we failed to detectasignificantincreaseinphosphorylationofthreonine-308 in the nucleus To monitor that nuclear Akt/PKB is activated after IR, we determined the capacity of nuclear and cytoplasmic Akt/PKB to phosphorylate bacterially expressed and purified GST-GSK-3 . Surprisingly, cytoplasmic Akt/PKB washardlyactivatedinresponsetoIRwhereasnuclearAkt/PKB phosphorylated GSK-3 very efficiently The obser- vation that nuclear Akt/PKB is activated upon IR almost exclu- sively suggests that all active Akt/PKB translocated into the nucleusor,evenmorelikely,thatactivationofAkt/PKBafterIR occurred within the nuclear compartment. In this context, phosphorylation of Mdm2 at serine-166 and serine-186 by Akt/PKB, which leads to nuclear translocation of Mdm2, can be disregarded
a)
In addition to the Akt/GSK-3 pathway described above, signaling through the Wnt pathway has been inferred to be of clinical relevance in schizophrenia. Members of the Wnt protein family are secreted glycoproteins implicated in diverse neuronal processes including brain development, regulation of synaptogenesis and synapse specificity, as well as in oncogenesis and determination of cell fateDopamine D2 receptor antagonism is a unifying property of all antipsychotic drugs in clinical use. Remarkably, the effector molecules through which these medications exert their actions remain poorly characterized. Increasing attention is being focused on Akt/glycogen synthase kinase-3 (GSK-3) and wingless (Wnt) signaling pathways which have been associated with schizophrenia in a number of genetic and postmortem studies. Antipsychotic medications may treat symptoms of psychosis, at least in part, through modulation of levels and activity of Akt, GSK-3 and Wnt-related intracellular signaling. The authors review evidence that Akt/GSK-3 and Wnt-related pathways are involved in the pathogenesis of schizophrenia as well as details of intracellular events related to these molecules mediated by both typical and atypical antipsychotic medications. Further study of Akt/GSK-3 and Wnt signaling may ultimately lead to alternative therapeutics of schizophrenia-related disorders. Wnt-signaling is a regulator of a number of endocrine functions in health and disease. Wnt signaling molecules are expressed in the human endocrine pancreas and regulate insulin secretion, crosstalk between adipocytes and endocrine cells and the proliferation of pancreas ?-cells. Wnt can bind to cell-surface receptors called frizzled and to lipoprotein receptor-related protein co-receptors (e.g. LRP5). Upon binding to frizzled and LFP5 co-receptors dishelved is activated and subsequently recruits the axin-APC-GSK3? complex to the cell membrane. The result is an inhibition of GSK3? phosphorylation of beta-catenin which then allows beta-catenin to form the bipartite beta-catenin/TCF transcription factor enhancing expression of Wnt target genes. Transcription factor 7
tors Antibodies Assay Kits GSK-3 Inhibitors Proteins/Enzymes Wnt/beta-Catenin Signaling Pathway ModulatorsRelated Questions
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