Publications by authors named "Nicolas Fossat"

26 Publications

  • Page 1 of 1

TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation.

Elife 2021 Feb 8;10. Epub 2021 Feb 8.

Embryology Unit, Children's Medical Research Institute, The University of Sydney, Sydney, Australia.

Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labeling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cells (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal differentiation of NCCs and compromised craniofacial tissue patterning. Following NCC delamination, low level of TWIST1-CRM activity is instrumental to stabilize the early NCC signatures and migratory potential by repressing the neural stem cell programs. High level of TWIST1 module activity at later phases commits the cells to the ectomesenchyme. Our study further revealed the functional interdependency of TWIST1 and potential neurocristopathy factors in NCC development.
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http://dx.doi.org/10.7554/eLife.62873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968925PMC
February 2021

Evolutionary selection of pestivirus variants with altered or no microRNA dependency.

Nucleic Acids Res 2020 06;48(10):5555-5571

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre 2650, Denmark.

Host microRNA (miRNA) dependency is a hallmark of the human pathogen hepatitis C virus (HCV) and was also described for the related pestiviruses, which are important livestock pathogens. The liver-specific miR-122 binds within the HCV 5' untranslated region (UTR), whereas the broadly expressed let-7 and miR-17 families bind two sites (S1 and S2, respectively) in the pestiviral 3' UTR. Here, we dissected the mechanism of miRNA dependency of the pestivirus bovine viral diarrhea virus (BVDV). Argonaute 2 (AGO2) and miR-17 binding were essential for viral replication, whereas let-7 binding was mainly required for full translational efficiency. Furthermore, using seed site randomized genomes and evolutionary selection experiments, we found that tropism could be redirected to different miRNAs. AGO cross-linking and immunoprecipitation (CLIP) experiments and miRNA antagonism demonstrated that these alternative variants bound and depended on the corresponding miRNAs. Interestingly, we also identified miRNA-independent variants that were obtained through acquisition of compensatory mutations near the genomic 3' terminus. Rescue experiments demonstrated that miRNA binding and 3' mutagenesis contribute to replication through mutually exclusive mechanisms. Altogether, our findings suggest that pestiviruses, although capable of miRNA-independent replication, took advantage of miRNAs as essential host factors, suggesting a favorable path during evolutionary adaptation.
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http://dx.doi.org/10.1093/nar/gkaa300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261151PMC
June 2020

TWIST1 Homodimers and Heterodimers Orchestrate Lineage-Specific Differentiation.

Mol Cell Biol 2020 05 14;40(11). Epub 2020 May 14.

Embryology Unit, Children's Medical Research Institute, The University of Sydney, Sydney, Australia.

The extensive array of basic helix-loop-helix (bHLH) transcription factors and their combinations as dimers underpin the diversity of molecular function required for cell type specification during embryogenesis. The bHLH factor TWIST1 plays pleiotropic roles during development. However, which combinations of TWIST1 dimers are involved and what impact each dimer imposes on the gene regulation network controlled by TWIST1 remain elusive. In this work, proteomic profiling of human TWIST1-expressing cell lines and transcriptome analysis of mouse cranial mesenchyme have revealed that TWIST1 homodimers and heterodimers with TCF3, TCF4, and TCF12 E-proteins are the predominant dimer combinations. Disease-causing mutations in TWIST1 can impact dimer formation or shift the balance of different types of TWIST1 dimers in the cell, which may underpin the defective differentiation of the craniofacial mesenchyme. Functional analyses of the loss and gain of TWIST1-E-protein dimer activity have revealed previously unappreciated roles in guiding lineage differentiation of embryonic stem cells: TWIST1-E-protein heterodimers activate the differentiation of mesoderm and neural crest cells, which is accompanied by the epithelial-to-mesenchymal transition. At the same time, TWIST1 homodimers maintain the stem cells in a progenitor state and block entry to the endoderm lineage.
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http://dx.doi.org/10.1128/MCB.00663-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225560PMC
May 2020

Ularcirc: visualization and enhanced analysis of circular RNAs via back and canonical forward splicing.

Nucleic Acids Res 2019 11;47(20):e123

Victor Chang Cardiac Research Institute.

Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc.
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http://dx.doi.org/10.1093/nar/gkz718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6846653PMC
November 2019

Dynamics of Wnt activity on the acquisition of ectoderm potency in epiblast stem cells.

Development 2019 04 5;146(7). Epub 2019 Apr 5.

Embryology Unit, Children's Medical Research Institute, Westmead, NSW 2145, Australia.

During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.
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http://dx.doi.org/10.1242/dev.172858DOI Listing
April 2019

Functional Interplay between RNA Viruses and Non-Coding RNA in Mammals.

Noncoding RNA 2019 Jan 14;5(1). Epub 2019 Jan 14.

Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.

Exploring virus⁻host interactions is key to understand mechanisms regulating the viral replicative cycle and any pathological outcomes associated with infection. Whereas interactions at the protein level are well explored, RNA interactions are less so. Novel sequencing methodologies have helped uncover the importance of RNA⁻protein and RNA⁻RNA interactions during infection. In addition to messenger RNAs (mRNAs), mammalian cells express a great number of regulatory non-coding RNAs, some of which are crucial for regulation of the immune system whereas others are utilized by viruses. It is thus becoming increasingly clear that RNA interactions play important roles for both sides in the arms race between virus and host. With the emerging field of RNA therapeutics, such interactions are promising antiviral targets. In this review, we discuss direct and indirect RNA interactions occurring between RNA viruses or retroviruses and host non-coding transcripts upon infection. In addition, we review RNA virus derived non-coding RNAs affecting immunological and metabolic pathways of the host cell typically to provide an advantage to the virus. The relatively few known examples of virus⁻host RNA interactions suggest that many more await discovery.
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http://dx.doi.org/10.3390/ncrna5010007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468702PMC
January 2019

A gene regulatory network anchored by LIM homeobox 1 for embryonic head development.

Genesis 2018 09 14;56(9):e23246. Epub 2018 Sep 14.

Embryology Unit, Children's Medical Research Institute, The University of Sydney, Sydney, New South Wales, Australia.

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1-anchored gene regulatory network. Using a gain-of-function approach, we identified genes that immediately respond to Lhx1 activation. Meta-analysis of the datasets of LHX1-responsive genes and genes expressed in the anterior tissues of mouse embryos at head-fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP-seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.
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http://dx.doi.org/10.1002/dvg.23246DOI Listing
September 2018

Formation of the Embryonic Head in the Mouse: Attributes of a Gene Regulatory Network.

Curr Top Dev Biol 2016 15;117:497-521. Epub 2016 Feb 15.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia; Systems Biology Institute Australia, Monash University, Clayton, Victoria, Australia.

The embryonic head is the first major body part to be constructed during embryogenesis. The allocation and the assembly of the progenitor tissues, which start at gastrulation, are accompanied by the spatiotemporal activity of transcription factors and signaling pathways that drives lineage specification, germ layer formation, and cell/tissue movement. The morphogenesis, regionalization, and patterning of the brain and craniofacial structures rely on the function of LIM-domain, homeodomain, and basic helix-loop-helix transcription factors. These factors constitute the central nodes of a gene regulatory network (GRN) which encompasses and intersects with signaling pathways involved with head formation. It is predicted that the functional output of this "head GRN" impacts on cellular function and cell-cell interactions that are essential for lineage differentiation and tissue modeling, which are key processes underpinning the formation of the head.
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http://dx.doi.org/10.1016/bs.ctdb.2015.11.019DOI Listing
December 2016

Conditional restoration and inactivation of Rbm47 reveal its tissue-context requirement for viability and growth.

Genesis 2016 Mar 5;54(3):115-22. Epub 2016 Feb 5.

Embryology Unit, Children's Medical Research Institute, Westmead, New South Wales, 2145, Australia.

Rbm47 encodes a RNA binding protein that is necessary for Cytidine to Uridine RNA editing. Rbm47(gt/gt) mutant mice that harbor inactivated Rbm47 display poor viability. Here it was determined that the loss of Rbm47(gt/gt) offspring is due to embryonic lethality at mid-gestation. It was further showed that growth of the surviving Rbm47(gt/gt) mutants is impaired. Rbm47 is expressed in both the visceral endoderm and the definitive endoderm. Using the utility of the switchable FlEx gene-trap cassette and the activity of Cre and FLP recombinases to generate mice that conditionally inactivate and restore Rbm47 function in tissue-specific manner, it was demonstrated that Rbm47 function is required in the embryo proper, and not the visceral endoderm, for viability and growth. genesis 54:115-122, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvg.22920DOI Listing
March 2016

Generation of genome-edited mouse epiblast stem cells via a detour through ES cell-chimeras.

Differentiation 2016 Apr-Jun;91(4-5):119-25. Epub 2015 Nov 21.

Embryology Unit, Children's Medical Research Institute, NSW 2145, Australia; Discipline of Medicine, Sydney Medical School, University of Sydney, NSW 2006, Australia. Electronic address:

Conventionally, mouse epiblast stem cells (EpiSCs) are derived directly from the epiblast or ectoderm germ layer of the post-implantation embryo. Self-renewing and multipotent EpiSC-like stem cells can also be derived by the conversion of embryonic stem cells (ESCs) via the provision of culture conditions that enable the maintenance of the EpiSCs. Here, we outline an experimental procedure for deriving EpiSCs from post-implantation chimeric embryos that are generated using genome-edited ESCs. This strategy enables the production of EpiSCs where (i) no genetically modified animals or ESCs are available, (ii) the impact of the genetic modification on post-implantation development, which may influence the property of the EpiSCs, is requisite knowledge for using the EpiSC for a specific investigation, and (iii) multiple editing of the genome is desirable to modify the biological attributes of the EpiSCs for studying, for example, the gene network activity on the trajectory of lineage differentiation and tissue morphogenesis.
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http://dx.doi.org/10.1016/j.diff.2015.10.004DOI Listing
September 2017

Context-specific function of the LIM homeobox 1 transcription factor in head formation of the mouse embryo.

Development 2015 Jun 14;142(11):2069-79. Epub 2015 May 14.

Embryology Unit, Children's Medical Research Institute, Westmead, New South Wales 2145, Australia Discipline of Medicine, Sydney Medical School, University of Sydney, Sydney, New South Wales 2006, Australia.

Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity.
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http://dx.doi.org/10.1242/dev.120907DOI Listing
June 2015

Re-editing the paradigm of Cytidine (C) to Uridine (U) RNA editing.

RNA Biol 2014 ;11(10):1233-7

a Embryology Unit; Children's Medical Research Institute ; Westmead , Australia.

Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing.
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http://dx.doi.org/10.1080/15476286.2014.996054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615559PMC
September 2015

Head formation: OTX2 regulates Dkk1 and Lhx1 activity in the anterior mesendoderm.

Development 2014 Oct 17;141(20):3859-67. Epub 2014 Sep 17.

Embryology Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia Discipline of Medicine, Sydney Medical School, University of Sydney, New South Wales 2006, Australia.

The Otx2 gene encodes a paired-type homeobox transcription factor that is essential for the induction and the patterning of the anterior structures in the mouse embryo. Otx2 knockout embryos fail to form a head. Whereas previous studies have shown that Otx2 is required in the anterior visceral endoderm and the anterior neuroectoderm for head formation, its role in the anterior mesendoderm (AME) has not been assessed specifically. Here, we show that tissue-specific ablation of Otx2 in the AME phenocopies the truncation of the embryonic head of the Otx2 null mutant. Expression of Dkk1 and Lhx1, two genes that are also essential for head formation, is disrupted in the AME of the conditional Otx2-deficient embryos. Consistent with the fact that Dkk1 is a direct target of OTX2, we showed that OTX2 can interact with the H1 regulatory region of Dkk1 to activate its expression. Cross-species comparative analysis, RT-qPCR, ChIP-qPCR and luciferase assays have revealed two conserved regions in the Lhx1 locus to which OTX2 can bind to activate Lhx1 expression. Abnormal development of the embryonic head in Otx2;Lhx1 and Otx2;Dkk1 compound mutant embryos highlights the functional intersection of Otx2, Dkk1 and Lhx1 in the AME for head formation.
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http://dx.doi.org/10.1242/dev.114900DOI Listing
October 2014

C to U RNA editing mediated by APOBEC1 requires RNA-binding protein RBM47.

EMBO Rep 2014 Aug 10;15(8):903-10. Epub 2014 Jun 10.

Embryology Unit, Children's Medical Research Institute, Westmead, NSW, Australia Discipline of Medicine, Sydney Medical School University of Sydney, Sydney, NSW, Australia.

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing.
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http://dx.doi.org/10.15252/embr.201438450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197048PMC
August 2014

Purkinje cells and Bergmann glia are primary targets of the TRα1 thyroid hormone receptor during mouse cerebellum postnatal development.

Development 2014 Jan;141(1):166-75

Université de Lyon, CNRS, INRA, Université Claude Bernard Lyon 1, École Normale Supérieure de Lyon, Institut de Génomique Fonctionnelle de Lyon, F-69364 Lyon, Cedex 07, France.

Thyroid hormone is necessary for normal development of the central nervous system, as shown by the severe mental retardation syndrome affecting hypothyroid patients with low levels of active thyroid hormone. The postnatal defects observed in hypothyroid mouse cerebellum are recapitulated in mice heterozygous for a dominant-negative mutation of Thra, the gene encoding the ubiquitous TRα1 receptor. Using CRE/loxP-mediated conditional expression approach, we found that this mutation primarily alters the differentiation of Purkinje cells and Bergmann glia, two cerebellum-specific cell types. These primary defects indirectly affect cerebellum development in a global manner. Notably, the inward migration and terminal differentiation of granule cell precursors is impaired. Therefore, despite the broad distribution of its receptors, thyroid hormone targets few cell types that exert a predominant role in the network of cellular interactions that govern normal cerebellum maturation.
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http://dx.doi.org/10.1242/dev.103226DOI Listing
January 2014

Wnt signalling in mouse gastrulation and anterior development: new players in the pathway and signal output.

Curr Opin Genet Dev 2013 Aug 19;23(4):454-60. Epub 2013 Apr 19.

Early Mammalian Development Laboratory, Research School of Biology, College of Medicine, Biology and Environment, Australian National University, Canberra, ACT, Australia.

Embryonic development and adult homeostasis are dependent upon the coordinated action of signal transduction pathways such as the Wnt signalling pathway which is used iteratively during these processes. In the early post-implantation mouse embryo, Wnt/β-catenin signalling activity plays a critical role in the formation of the primitive streak, progression of gastrulation and tissue patterning in the anterior-posterior axis. The net output of the signalling pathway is influenced by the delivery and post-translational modification of the ligands, the counteracting activities of the activating components and the negative modulators, and the molecular interaction of β-catenin, TCF and other factors regulating the transcription of downstream target genes.
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http://dx.doi.org/10.1016/j.gde.2013.03.001DOI Listing
August 2013

Modulation of WNT signaling activity is key to the formation of the embryonic head.

Cell Cycle 2012 Jan 1;11(1):26-32. Epub 2012 Jan 1.

Embryology Unit, Children's Medical Research Institute, University of Sydney, Westmead, Australia.

The formation of the embryonic head begins with the assembly of the progenitor tissues of the brain, the head and face primordia and the foregut that are derived from the primary germ layers during gastrulation. Specification of the anterior-posterior polarity of major body parts and the morphogenesis of the head and brain specifically is driven by inductive signals including those mediated by BMP, Nodal, FGF and WNT. A critical role of β-catenin dependent WNT signalling activity for head morphogenesis has been revealed through the analysis of the phenotypic impact of loss of function mutation of an antagonist: DKK1, a transcriptional repressor: GSC; and the outcome of interaction of Dkk1 with genes coding three components of the canonical signalling pathway: the ligand WNT3, the co-receptor LRP6 and the transcriptional co-factor, β-catenin. The findings highlight the requirement of a stringent control of the timing, domain and level of canonical WNT signalling activity for the formation of the embryonic head.
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http://dx.doi.org/10.4161/cc.11.1.18700DOI Listing
January 2012

Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm.

Development 2011 Oct 8;138(20):4511-22. Epub 2011 Sep 8.

Embryology Unit, Children's Medical Research Institute, Westmead, NSW 2145, Australia.

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.
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http://dx.doi.org/10.1242/dev.063867DOI Listing
October 2011

Stringent requirement of a proper level of canonical WNT signalling activity for head formation in mouse embryo.

Development 2011 Feb 12;138(4):667-76. Epub 2011 Jan 12.

Embryology Unit, Children's Medical Research Institute, University of Sydney, 214 Hawkesbury Road, Westmead, Sydney, NSW 2145, Australia.

In mouse embryos, loss of Dickkopf-1 (DKK1) activity is associated with an ectopic activation of WNT signalling responses in the precursors of the craniofacial structures and leads to a complete truncation of the head at early organogenesis. Here, we show that ENU-induced mutations of genes coding for two WNT canonical pathway factors, the co-receptor LRP6 and the transcriptional co-activator β-catenin, also elicit an ectopic signalling response and result in loss of the rostral tissues of the forebrain. Compound mutant embryos harbouring combinations of mutant alleles of Lrp6, Ctnnb1 and Dkk1 recapitulate the partial to complete head truncation phenotype of individual homozygous mutants. The demonstration of a synergistic interaction of Dkk1, Lrp6 and Ctnnb1 provides compelling evidence supporting the concepts that (1) stringent regulation of the level of canonical WNT signalling is necessary for head formation, (2) activity of the canonical pathway is sufficient to account for the phenotypic effects of mutations in three different components of the signal cascade and (3) rostral parts of the brain and the head are differentially more sensitive to canonical WNT signalling and their development is contingent on negative modulation of WNT signalling activity.
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http://dx.doi.org/10.1242/dev.052803DOI Listing
February 2011

Otx2 gene deletion in adult mouse retina induces rapid RPE dystrophy and slow photoreceptor degeneration.

PLoS One 2010 Jul 21;5(7):e11673. Epub 2010 Jul 21.

Institut de Génomique Fonctionnelle de Lyon, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Lyon, Université de Lyon, Lyon, France.

Background: Many developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells.

Methodology/principal Findings: Conditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease.

Conclusions: Our novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011673PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908139PMC
July 2010

A transcriptome landscape of mouse embryogenesis.

Dev Cell 2007 Dec;13(6):761-2

Embryology Unit, Children's Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville NSW 2145, Australia.

Coordinated regulation of genetic activity underpins formation of the body plan and morphogenesis of embryonic structures. In this issue of Developmental Cell, Mitiku and Baker describe a chronological series of transcriptomes of postimplantation mouse embryos at gastrulation and early organogenesis, providing a valuable resource for studying the dynamics of both genome-wide and gene-specific transcriptional activities that accompany mouse embryogenesis.
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http://dx.doi.org/10.1016/j.devcel.2007.11.011DOI Listing
December 2007

A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors.

BMC Dev Biol 2007 Nov 2;7:122. Epub 2007 Nov 2.

IGFL, UMR CNRS 5242-INRA 1237-ENS, IFR128 Lyon-Gerland, 46 allée d'Italie, 69364 Lyon Cedex 07, France.

Background: Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing.

Results: We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type.

Conclusion: The GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.
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http://dx.doi.org/10.1186/1471-213X-7-122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2204009PMC
November 2007

Temporal and spatial delineation of mouse Otx2 functions by conditional self-knockout.

EMBO Rep 2006 Aug 14;7(8):824-30. Epub 2006 Jul 14.

BMC, UMR CNRS 5161-INRA 1237-ENS, IFR128 Lyon-Gerland, 46 allée d'Italie, 69364 Lyon Cedex 07, France.

To identify the independent spatial and temporal activities of the essential developmental gene the Otx2, the germline mutation of which is lethal at embryonic day 8.5, we floxed one allele and substituted the other with an inducible CreER recombinase gene. This makes 'trans' self-knockout possible at any developmental stage. The transient action of tamoxifen pulses allows time-course mutation. We demonstrate efficient temporal knockout and demarcate spatio-temporal windows in which Otx2 controls the head, brain structures and body development.
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http://dx.doi.org/10.1038/sj.embor.7400751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1525150PMC
August 2006

Molecular dissection reveals decreased activity and not dominant negative effect in human OTX2 mutants.

J Mol Med (Berl) 2006 Jul 11;84(7):604-15. Epub 2006 Apr 11.

LBMC, ENS-Lyon, IFR128 Lyon-Gerland, 46 allée d'Italie, 69364, Lyon, Cedex 07, France.

The paired-type homeodomain transcription factor Otx2 is essential for forebrain and eye development. Severe ocular malformations in humans have recently been associated with heterozygous OTX2 mutations. To document the molecular defects in human mutants, Otx2 structural characterization was carried out. A collection of deletion and point mutants was created to perform transactivation, DNA binding, and subcellular localization analyses. Transactivation was ascribed to both N- and C-termini of the protein, and DNA binding to the minimal homeodomain, where critical amino acid residues were identified. Acute nuclear localization appeared controlled by a nuclear localization sequence located within the homeodomain which acts in conjunction with a novel nuclear retention domain that we unraveled located in the central part of the protein. This region, which is poorly conserved among Otx proteins, was also endowed with dominant negative activity suggesting that it might confer unique properties to Otx2. Molecular diagnostic of human mutant OTX2 proteins discriminates hypomorphic and loss of function mutations from other mutations that may not be relevant to ocular pathology.
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http://dx.doi.org/10.1007/s00109-006-0048-2DOI Listing
July 2006

Alternative usage of Otx2 promoters during mouse development.

Dev Dyn 2005 May;233(1):154-60

LBMC, ENS-Lyon, IFR128 Lyon-Gerland, 69364 Lyon Cedex 07, France.

Our previous structural analysis of mouse Otx2 transcripts has revealed the existence of three different promoters and suggested that the corresponding mRNAs could exhibit specific expression patterns. Here, we analyze the precise dynamics of their expression throughout mouse development. Their spatial distribution was determined by isoform-specific in situ hybridization and their relative abundance by real-time reverse transcriptase-polymerase chain reaction. Although the three promoters may be used in the same areas, we show that transcription preferentially occurs from the proximal promoter at onset of gene activity in early embryogenesis, and switches to the more distal one in most of the sites of expression in the adult brain. During gestation, their relative utilization becomes inverted. The third promoter, which shows no activity in embryonic stem cells and is moderately expressed during embryogenesis, is mostly used in specific areas derived from the rostral part of the neural tube.
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http://dx.doi.org/10.1002/dvdy.20287DOI Listing
May 2005
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