Point Mutations in GLI3 Lead to Misregulation of its Subcellular Localization

Background Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. Principal Findings We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-α4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. Conclusions The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations

Differential Coupling of Self-Renewal Signaling Pathways in Murine Induced Pluripotent Stem Cells

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors

An archaeal compound as a driver of Parkinson’s disease pathogenesis

Patients with Parkinson’s disease (PD) exhibit differences in their gut microbiomes compared to healthy individuals. Although differences have most commonly been described in the abundances of bacterial taxa, changes to viral and archaeal populations have also been observed. Mechanistic links between gut microbes and PD pathogenesis remain elusive but could involve molecules that promote α-synuclein aggregation. Here, we show that 2-hydroxypyridine (2-HP) represents a key molecule for the pathogenesis of PD. We observe significantly elevated 2-HP levels in faecal samples from patients with PD or its prodrome, idiopathic REM sleep behaviour disorder (iRBD), compared to healthy controls. 2-HP is correlated with the archaeal species Methanobrevibacter smithii and with genes involved in methane metabolism, and it is detectable in isolate cultures of M. smithii. We demonstrate that 2-HP is selectively toxic to transgenic α-synuclein overexpressing yeast and increases α-synuclein aggregation in a yeast model as well as in human induced pluripotent stem cell derived enteric neurons. It also exacerbates PD-related motor symptoms, α-synuclein aggregation, and striatal degeneration when injected intrastriatally in transgenic mice overexpressing human α-synuclein. Our results highlight the effect of an archaeal molecule in relation to the gut-brain axis, which is critical for the diagnosis, prognosis, and treatment of PD.

Expression of Trps1 during mouse embryonic development

The Trps1 gene codes for an atypical member of the GATA type family of transcription factors. Mutations in human TRPS1 lead to the tricho-rhino-phalangeal syndrome types I and III, which are characterized by craniofacial and skeletal abnormalities and disturbed hair development. Correspondingly, during mouse embryonic development strong Trps1 expression is found in the cartilage condensations, the developing joints, the hair follicles and in the developing snout. In addition, Trps1 is expressed surrounding the skeletal condensations, in the trachea, the intervertebral disks, and in lung and gut mesenchyme. A complex pattern of expression is also found in the developing brain

Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification

Exostosin1 (Ext1) belongs to a family of glycosyltransferases necessary for the synthesis of the heparan sulfate (HS) chains of proteoglycans, which regulate signaling of several growth factors. Loss of tout velu (ttv), the homolog of Ext1 in Drosophila, inhibits Hedgehog movement. In contrast, we show that reduced HS synthesis in mice carrying a hypomorphic mutation in Ext1 results in an elevated range of Indian hedgehog (Ihh) signaling during embryonic chondrocyte differentiation. Our data suggest a dual function for HS: First, HS is necessary to bind Hedgehog in the extracellular space. Second, HS negatively regulates the range of Hedgehog signaling in a concentration-dependent manner. Additionally, our data indicate that Ihh acts as a long-range morphogen, directly activating the expression of parathyroid hormone-like hormone. Finally, we propose that the development of exostoses in the human Hereditary Multiple Exostoses syndrome can be attributed to activation of Ihh signaling

Ext1 regulates chondrocyte differentiation

Hereditary multiple exostoses (HME) syndrome is an autosomal dominant inherited human disorder, which is characterized by the formation of multiple cartilaginous capped benign tumors (exostoses) that develop from the growth plate of endochondral bones. So far HME has been linked to missense or frameshift mutations in the tumor suppressor genes Ext1 and Ext2. Both are glycosyltransferases involved in heparan sulfate (HS) biosynthesis. It has been shown that in Drosophila the homolog of Ext1, tout velu (ttv), is required for transport of hedgehog (Hh). One of the vertebrate homologs of Hh, Indian hedgehog (Ihh), is a key regulator of endochondral ossification. Ext1 knockout mice are embryonic lethal due to gastrulation defects. We are analyzing a mouse line carrying a hypomorphic allele of Ext1 obtained from a genetrap screen (Ext1-GT). Since Ext1 mutations lead to skeletal deformations in human patients we started to analyze the role of Ext1 and HS-chains during endochondral ossification focussing on the potential role of Ext1 in mediating the Ihh signal. Analysis of the Ext1-GT mutants revealed a severe delay in chondrocyte differentiation. We could show that less amounts of HS in these mutants allow further diffusion of Ihh whereas treatment of limb explants in a limb culture system with heparin leads to a restriction of the Ihh signal. These experiments implicate an important role of HS in establishing a gradient of Ihh signalling in cartilage thereby regulating chondrocyte differentiation

Alternative polyadenylation signals and promoters act in concert to control tissue-specific expression of the Opitz syndrome gene MID1

<p>Abstract</p> <p>Background</p> <p>Mutations in the X-linked <it>MID1 </it>gene are responsible for Opitz G/BBB syndrome, a malformation disorder of developing midline structures. Previous Northern blot analyses revealed the existence of at least three <it>MID1 </it>transcripts of differing lengths.</p> <p>Results</p> <p>Here we show that alternative polyadenylation generates the size differences observed in the Northern blot analyses. Analysis of EST data together with additional Northern blot analyses proved tissue-specific usage of the alternative polyadenylation sites. Bioinformatic characterization of the different 3'UTRs of <it>MID1 </it>revealed numerous RNA-protein interaction motifs, several of which turned out to be conserved between different species. Furthermore, our data suggest that mRNA termination at different polyadenylation sites is predetermined by the choice of alternative 5'UTRs and promoters of the <it>MID1 </it>gene, a mechanism that efficiently allows synergistic function of 5' and 3'UTRs.</p> <p>Conclusion</p> <p><it>MID1 </it>expression is tightly regulated through concerted action of alternative promoters and alternative polyadenylation signals both during embryonic development and in the adult.</p

Active transport of the Ubiquitin Ligase MID1 along the microtubules Is regulated by Protein Phosphatase 2A

Mutations in the MID1 protein have been found in patients with Opitz BBB/G syndrome (OS), which is characterised by multiple malformations of the ventral midline. MID1 is a microtubule-associated protein that stabilizes microtubules and, in association with the regulatory subunit of protein phosphatase 2A (PP2A), α4, provides ubiquitin ligase activity for the ubiquitin-specific modification of PP2A. Using Fluorescence Recovery After Photobleaching (FRAP) technology, we show here that MID1 is actively and bi-directionally transported along the microtubules, and that this movement is directly linked to its MAP kinase and PP2A-mediated phosphorylation status. Intact transport depends on both kinesins and dyneins and is inhibited upon colcemide treatments. MID1 proteins carrying missense mutations in the α4 binding domain still bind the microtubules but cannot be actively transported. Likewise, knock-down of the α4 protein, inhibition of PP2A activity by okadaic acid and fostriecin or the simulation of permanent phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP, while preserving its microtubule-association. In summary, our data uncover an unexpected and novel function for PP2A, its regulatory subunit α4 and PP2A/α4/mTOR signaling in the active transport of the MID1 ubiquitin ligase complex along the cytoskeleton. Furthermore, a failure in the microtubule directed transport of this protein complex would be an attractive mechanism underlying the pathogenesis of OS in patients with B-box1 mutations

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert\u27s membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo. © 2011 Elsevier Inc

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert\u27s membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo. © 2011 Elsevier Inc