Selected Manuscripts (pdf) & Abstracts


Nature Genetics. 2017

Am J Hum Genet. 2017 

Loss-of-Function Mutations in LGI4, a Secreted Ligand Involved in Schwann Cell 

Myelination, Are Responsible for Arthrogryposis Multiplex Congenita.

Xue S, Maluenda J, Marguet F, Shboul M, Quevarec L, Bonnard C, Ng AY, Tohari S, Tan TT, Kong MK, Monaghan KG, Cho MT, Siskind CE, Sampson JB, Rocha CT, Alkazaleh F, Gonzales M, Rigonnot L, Whalen S, Gut M, Gut I, Bucourt M, Venkatesh B, Laquerrière A, Reversade Bruno, Melki J

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through genetic mapping of disease loci and whole-exome sequencing in four unrelated multiplex families presenting with severe AMC, we identified biallelic loss-of-function mutations in LGI4 (leucine-rich glioma-inactivated 4). LGI4 is a ligand secreted by Schwann cells that regulates peripheral nerve myelination via its cognate receptor ADAM22 expressed by neurons. Immunolabeling experiments and transmission electron microscopy of the sciatic nerve from one of the affected individuals revealed a lack of myelin. Functional tests using affected individual-derived iPSCs showed that these germline mutations caused aberrant splicing of the endogenous LGI4 transcript and in a cell-based assay impaired the secretion of truncated LGI4 protein. This is consistent with previous studies reporting arthrogryposis in Lgi4-deficient mice due to peripheral hypomyelination. This study adds to the recent reports implicating defective axoglial function as a key cause of AMC.

De novo mutations in SMCHD1 cause Bosma arhinia microphthalmia syndrome
and abrogate nasal development.

Christopher T. Gordon, Shifeng Xue, Gökhan Yigit, Hicham Filali, Kelan Chen, Nadine Rosin, Koh-ichiro, Yoshiura, Myriam Oufadem, Tamara J. Beck, Ruth McGowan, Alex C. Magee, Janine Altmüller, Camille Dion, Holger Thiele, Alexandra D. Gurzau, Peter Nürnberg, Dieter Meschede, Wolfgang Mühlbauer, Nobuhiko Okamoto, Vinod Varghese, Rachel Irving, Sabine Sigaudy, Denise Williams, S. Faisal Ahmed, Carine Bonnard, Mung Kei Kong, Ilham Ratbi, Nawfal Fejjal, Meriem Fikri, Siham Chafai Elalaoui, Hallvard Reigstad, Christine Bole-Feysot, Patrick Nitschké, Nicola Ragge, Nicolas Lévy, Gökhan Tunçbilek, Audrey S.M. Teo, Michael L. Cunningham, Abdelaziz Sefiani, Hülya Kayserili, James M. Murphy, Chalermpong Chatdokmaiprai, Axel M. Hillmer, Duangrurdee Wattanasirichaigoon, Stanislas Lyonnet, Frédérique Magdinier, Asif Javed, Marnie E. Blewitt, Jeanne Amiel, Bernd Wollnik, Bruno Reversade

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.


CELL. 2016  

Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

Zhong FL, Mamaï O, Sborgi L, Boussofara L, Hopkins R, Robinson K, Szeverényi I, Takeichi T, Balaji R, Lau A, Tye H, Roy K, Bonnard C, Ahl PJ, Jones LA, Baker P, Lacina L, Otsuka A, Fournie PR, Malecaze F, Lane EB, Akiyama M, Kabashima K, Connolly JE, Masters SL, Soler VJ, Omar SS, McGrath JA, Nedelcu R, Gribaa M, Denguezli M, Saad A, Hiller S, Bruno Reversade 

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

eLiFE. 2016  

Quantitative imaging reveals real-time Pou5f3-Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish

Mireia Perez Camps , Jing Tian , Serene Chng, Kaiping Sem , Thankiah Sudhaharan, Cathleen Teh, Malte Wachsmuth , Vladimir Korzh , Sohail Ahmed, Bruno Reversade.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3-Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela/Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3-Nanog complexes and the activation of downstream BMP signalling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3-Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis.

Am J Hum Genet. 2016 

Loss-of-function mutations in ELMO2 cause intraosseous vascular malformation by impeding RAC1 signaling

Arda Cetinkaya, Jingwei Rachel Xiong, Ibrahim Vargel, Kemal Kosemehmetoglu, Halil Ibrahim Canter, Omer Faruk Gerdan,Nicola Longo, Ahmad Alzahrani, Mireia Perez Camps, Ekim Zihni Taskiran,Simone Laupheimer, Lorenzo D. Botto, Eeswari Paramalingam, Zeliha Gormez, Elif Uz, Bayram Yuksel, Sxevket Ruacan, Mahmut Sxamil Sagıroglu, Tokiharu Takahashi, Bruno Reversade, Nurten Ayse Akarsu

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Vascular malformations are non-neoplastic expansions of blood vessels that arise due to errors during angiogenesis. They are a heterogeneous group of sporadic or inherited vascular disorders characterized by localized lesions of arteriovenous, capillary, or lymphatic origin. Vascular malformations that occur inside bone tissue are rare. Herein, we report loss-of-function mutations in ELMO2 (which translates extracellular signals into cellular move-ments) that are causative for autosomal-recessive intraosseous vascular malformation (VMOS) in five different families. Individuals with VMOS suffer from life-threatening progressive expansion of the jaw, craniofacial, and other intramembranous bones caused by malformed blood vessels that lack a mature vascular smooth muscle layer. Analysis of primary fibroblasts from an affected individual showed that absence of ELMO2 correlated with a significant downregulation of binding partner DOCK1, resulting in deficient RAC1-dependent cell migration. Unexpectedly, elmo2-knockout zebrafish appeared phenotypically normal, suggesting that there might be human-specific ELMO2 requirements in bone vasculature homeostasis or genetic compensation by related genes. Comparative phylogenetic analysis indicated that elmo2 originated upon the appearance of intramembranous bones and the jaw in ancestral vertebrates, implying that elmo2 might have been involved in the evolution of these novel traits. The present findings highlight the necessity of ELMO2 for maintaining vascular integrity, specifically in intramembranous bones.

Am J Hum Genet. 2016 

De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations.

Reijnders MR, Zachariadis V, Latour B, Jolly L, Mancini GM, Pfundt R, Wu KM, van Ravenswaaij-Arts CM, Veenstra-Knol HE, Anderlid BM, Wood SA, Cheung SW, Barnicoat A, Probst F, Magoulas P, Brooks AS, Malmgren H, Harila-Saari A, Marcelis CM, Vreeburg M, Hobson E, Sutton VR, Stark Z, Vogt J, Cooper N, Lim JY, Price S, Lai AH, Domingo D, Bruno Reversade ; DDD Study, Gecz J, Gilissen C, Brunner HG, Kini U, Roepman R, Nordgren A, Kleefstra T.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Academic Medical Centre & VU University Medical Center, Reproductive Biology Laboratory, Amsterdam, the Netherlands.

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function.


Current Biology. 2015                                                                                    

Gmnc Is a Master Regulator of the Multiciliated Cell Differentiation Program

Feng Zhou, Vijay Narasimhan, Mohammad Shboul, Yan Ling Chong, Bruno Reversade, Sudipto Roy

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Multiciliated cells (MCCs) differentiate hundreds of motile cilia that generate mechanical force required to drive fluid movement over epithelia [1 and 2]. For example, metachronal beating of MCC cilia in the mammalian airways clears mucus that traps inhaled pathogens and pollutants. Consequently, abnormalities in MCC differentiation or ciliary motility have been linked to an expanding spectrum of human airway diseases [3, 4,5 and 6]. The current view posits that MCC precursors are singled out by the inhibition of Notch signaling. MCC precursors then support an explosive production of basal bodies, which migrate to the apical surface, dock with the plasma membrane, and seed the growth of multiple motile cilia. At the center of this elaborate differentiation program resides the coiled-coil-containing protein Multicilin, which transcriptionally activates genes for basal body production and the gene for FoxJ1, the master regulator for basal body docking, cilia formation, and motility [7 and 8]. Here, using genetic analysis in the zebrafish embryo, we discovered that Gmnc is a novel determinant of the MCC fate. Like Multicilin, Gmnc is a coiled-coil-containing protein of the Geminin family. We show that Gmnc functions downstream of Notch signaling, but upstream of Multicilin in the developmental pathway controlling MCC specification. Moreover, we find that loss of Gmnc in Xenopus embryos also causes loss of MCC differentiation and that overexpression of the protein is sufficient to induce supernumerary MCCs. Together, our data identify Gmnc as an evolutionarily conserved master regulator functioning at the top of the hierarchy of transcription factors involved in MCC differentiation.

Cell Stem Cell. 2015                                                                          

ELABELA Is an Endogenous Growth Factor that Sustains hESC Self-Renewal 

via the PI3K/AKT Pathway

Lena Ho, Shawn Y.X. Tan, Sheena Wee, Yixuan Wu, Sam J.C. Tan, Navin B. Ramakrishna, Serene C. Chng, Srikanth Nama, Iwona Sczerbineska, Winston Chan, Stuart Avery, Norihiro Tsuneyoshi, Huck Hui Ng, Jayantha Gunaratne, N. Ray Dunn, Bruno Reversade

Institute of Medical Biology, A*STAR, Singapore, Singapore.

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFβ pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.

Am J Hum Genet.2015 

Recurrent De Novo Mutations Affecting Residue Arg138 of Pyrroline-5-Carboxylate

Synthase Cause a Progeroid Form of Autosomal-Dominant Cutis Laxa.

Björn Fischer-Zirnsak, Nathalie Escande-Beillar, Jaya Ganesh, Yu Xuan Tan, Mohammed Al Bughaili, Angela E. Lin, Inderneel Sahai, Paulina Bahena, Sara L. Reichert, Abigail Loh, Graham D. Wright, Jaron Liu, Elisa Rahikkala, Eniko K. Pivnick, Asim F. Choudhri, Ulrike Krüger, Tomasz Zemojtel, Conny van Ravenswaaij-Arts, Roya Mostafavi, Irene Stolte-Dijkstra, Sofie Symoens, Leila Pajunen, Lihadh Al-Gazali, David Meierhofer, Peter N. Robinson, Stefan Mundlos, Camilo E. Villarroel, Peter Byers, Amira Masri, Stephen P. Robertson, Ulrike Schwarze, Bert Callewaert, Bruno Reversade, Uwe Kornak

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Progeroid disorders overlapping with De Barsy syndrome (DBS) are collectively denoted as autosomal-recessive cutis laxa type 3 (ARCL3). They are caused by biallelic mutations in PYCR1 or ALDH18A1, encoding pyrroline-5-carboxylate reductase 1 and pyrroline-5-carboxylate synthase (P5CS), respectively, which both operate in the mitochondrial proline cycle. We report here on eight unrelated individuals born to non-consanguineous families clinically diagnosed with DBS or wrinkly skin syndrome. We found three heterozygous mutations in ALDH18A1 leading to amino acid substitutions of the same highly conserved residue, Arg138 in P5CS. A de novo origin was confirmed in all six probands for whom parental DNA was available. Using fibroblasts from affected individuals and heterologous overexpression, we found that the P5CS-p.Arg138Trp protein was stable and able to interact with wild-type P5CS but showed an altered sub-mitochondrial distribution. A reduced size upon native gel electrophoresis indicated an alteration of the structure or composition of P5CS mutant complex. Furthermore, we found that the mutant cells had a reduced P5CS enzymatic activity leading to a delayed proline accumulation. In summary, recurrent de novo mutations, affecting the highly conserved residue Arg138 of P5CS, cause an autosomal-dominant form of cutis laxa with progeroid features. Our data provide insights into the etiology of cutis laxa diseases and will have immediate impact on diagnostics and genetic counseling.

eLiFE. 2015 May 27. 

The hormonal peptide Elabela guides angioblasts to the midline

during vasculogenesis

 Helker C. S. M.,  Schuermann A, Pollmann C , Chng S C, Kiefer F ,  Reversade B, Herzog W 

Institute of Medical Biology, A*STAR, Singapore, Singapore.

A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor (Vegf) has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplnr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis.

Human Molecular Genetics. 2015 Feb 24. 

Loss of the Scavenger mRNA Decapping Enzyme DCPS Causes Syndromic Intellectual Disability with Neuromuscular Defects.

Ng C, Shboul M, Taverniti V, Bonnard C, Lee H, Eskin A, Nelson SF, Al-Raqad M, Altawalbeh S, Séraphin B, Reversade B.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

mRNA decay is an essential and active process that allows cells to continuously adapt gene expression to internal and environmental cues. There are two mRNA degradation pathways: 3' to 5' and 5' to 3'. The DCPS protein is the scavenger mRNA decapping enzyme which functions in the last step of the 3' end mRNA decay pathway. We have identified a DCPS pathogenic mutation in a large family with three affected individuals presenting with a novel recessive syndrome consisting of craniofacial anomalies, intellectual disability and neuromuscular defects. Using patient's primary cells, we show that this homozygous splice mutation results in a DCPS loss-of-function allele. Diagnostic biochemical analyses using various m7G cap derivatives as substrates reveal no DCPS enzymatic activity in patient's cells. Our results implicate DCPS and more generally RNA catabolism, as a critical cellular process for neurological development, normal cognition and organismal homeostasis in humans.


Neuron. 2014 Dec 22. 

Katanin p80 Regulates Human Cortical Development by Limiting Centriole and Cilia Number.

Hu WF, Pomp O, Ben-Omran T, Kodani A, Henke K, Mochida GH, Yu TW, Woodworth MB, Bonnard C, Raj GS, Tan TT, Hamamy H, Masri A, Shboul M, Al Saffar M, Partlow JN, Al-Dosari M, Alazami A, Alowain M, Alkuraya FS, Reiter JF, Harris MP, Reversade B, Walsh CA.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.

Journal of Investigative Dermatology. 2014 Jul 22. 

Multiple Self-Healing Palmoplantar Carcinoma: A Familial Predisposition to Skin Cancer with Primary Palmoplantar and Conjunctival Lesions.

Mamaï O, Boussofara L, Denguezli M, Escande-Beillard N, Kraeim W, Merriman B, Ben Charfeddine I, Stevanin G, Bouraoui S, Amara A, Mili A, Nouira R, H'mida D, Sriha B, Gribaa M, Saad A, Reversade B.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Familial keratoacanthomas (KAs) are characterized by the appearance of multiple epithelial tumors that are believed to arise from adjoining hair follicles (Schwartz, 1994). These lesions, which phenotypically and histologically resemble squamous cell carcinomas (SCCs) (Cribier et al., 1999), have a fast evolution and spontaneous regression. To date, four familial forms of multiple KAs have been described: (1) multiple self-healing squamous epithelioma (MSSE; Smith, 1948); (2) the Grzybowski syndrome (Grzybowski, 1950); (3) the Witten–Zak syndrome (Witten and Zak, 1952); and 4) the Muir-Torre syndrome (Muir et al., 1967). The Muir-Torre syndrome (MIM#158320) is caused by mutations in the DNA-repair genes MSH2/MLH1 (Bapat et al., 1996; Kruse et al., 1998), and MSSE (MIM#132800) is due to mutations in TGFBR1 (Goudie et al., 2011), but the causative genes underlying the other two familial KAs are still not known. We describe here, in a five-generation Tunisian family comprising 27 affected individuals with autosomal dominant transmission of palmoplantar KAs, an unreported syndrome that we have named multiple self-healing palmoplantar carcinoma MSPC. MSPC has the distinctive feature of mainly affecting epithelial tissues that are devoid of hair follicles, such as palmoplantar skin and the conjunctival epithelium.


Developmental Cell. 2013 Dec;(27):1-9.                                  

ELABELA: A Hormone Essential for Heart Development Signals via the Apelin Receptor

Chng SHo L, Tian JReversade B

Institute of Medical Biology, A*STAR, Singapore, Singapore.

We report here the discovery and characterization of a gene, ELABELA (ELA), encoding a conserved hormone of 32 amino acids. Present in human embryonic stem cells, ELA is expressed at the onset of zebrafish zygotic transcription and is ubiquitous in the naive ectodermal cells of the embryo. Using zinc-finger-nuclease-mediated gene inactivation in zebrafish, we created an allelic series of ela mutants. ela null embryos have impaired endoderm differentiation potential marked by reduced gata5 and sox17 expression. Loss of Ela causes embryos to develop with a rudimentary heart or no heart at all, surprisingly phenocopying the loss of the apelin receptor (aplnr), which we show serves as Ela's cognate G protein-coupled receptor. Our results reveal the existence of a peptide hormone, ELA, which, together with APLNR, forms an essential signaling axis for early cardiovascular development.

Human Genetics. 2013 Nov 1. 

C5orf42 is the major gene responsible for OFD syndrome type VI.

Lopez E, Thauvin-Robinet C, Reversade B, Khartoufi NE, Devisme L, Holder M, Ansart-Franquet H, Avila M, Lacombe D, Kleinfinger P, Kaori I, Takanashi JI, Le Merrer M, Martinovic J, Noël C, Shboul M, Ho L, Güven Y, Razavi F, Burglen L, Gigot N, Darmency-Stamboul V, Thevenon J, Aral B, Kayserili H, Huet F, Lyonnet S, Le Caignec C, Franco B, Rivière JB, Faivre L, Attié-Bitach T.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the "Joubert syndrome related disorders". Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies. 

Mol Biol Cell. 2013 July 31. 

Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the Integrator complex.

Jodoin JN, Sitaram P, Albrecht TR, May SB, Shboul M, Lee E, Reversade B, Wagner EJ, Lee LA.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

We previously reported that Asunder (ASUN) is essential for recruitment of dynein motors to the nuclear envelope (NE) and nucleus-centrosome coupling at the onset of cell division in cultured human cells and Drosophila spermatocytes, though the mechanisms underlying this regulation remain unknown. We have also recently identified ASUN as a functional component of Integrator (INT), a multi-subunit complex required for 3'-end processing of small nuclear RNAs (snRNAs). We now provide evidence that ASUN acts in the nucleus in concert with other INT components to mediate recruitment of dynein to the NE. Knockdown of other individual INT subunits in HeLa cells recapitulates the loss of perinuclear dynein in ASUN-siRNA cells. Forced localization of ASUN to the cytoplasm via mutation of its nuclear localization sequence (NLS) blocks its capacity to restore perinuclear dynein in both cultured human cells lacking ASUN and Drosophila asun spermatocytes. Additionally, the levels of several INT subunits are reduced at G2/M when dynein is recruited to the NE, suggesting that INT does not directly mediate this step. Taken together, our data support a model in which a nuclear INT complex promotes recruitment of cytoplasmic dynein to the NE, possibly via a mechanism involving RNA processing.

Development.  2013 Jan 14. 

Lim CY, Reversade B, Knowles BB, Solter D.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Cellular differentiation during embryogenesis involves complex gene regulation to enable the activation and repression of genes. Here, we show that mesodermal competence is inhibited in Xenopus embryos depleted of histones H3 and H3.3, which fail to respond to Nodal/Activin signaling and exhibit concomitant loss of mesodermal gene expression. We find that transcriptional activation in gastrula embryos does not correlate with promoter deposition of H3.3. Instead, gastrulation defects in H3.3/H3-deficient embryos are partially rescued with concurrent depletion of the linker histone H1A. In addition, we show that linker histone H1-induced premature loss of mesodermal competence in animal cap explants can be abrogated with the overexpression of nucleosomal H3.3/H3. Our findings establish a chromatin-mediated regulatory mechanism in which a threshold level of H3 is required to prevent H1-induced gene repression, and thus facilitate mesodermal differentiation in response to inductive signaling.


Nature Genetics. 2012 Nov;44(11):1272-6.

Pohler E, Mamai O, Hirst J, Zamiri M, Horn H, Nomura T, Irvine AD, Moran B, Wilson NJ, Smith FJ, Goh CS, Sandilands A, Cole C, Barton GJ, Evans AT, Shimizu H, Akiyama M, Suehiro M, Konohana I, Shboul M, Teissier S,Boussofara L, Denguezli M, Saad A, Gribaa M, Dopping-Hepenstal PJ, McGrath JA, Brown SJ, Goudie DR, Reversade B, Munro CS, McLean WH.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin-binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin-binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine  phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signalling and cellular proliferation.

Nature Genetics. 2012 May 13;44(6):703-13.

Mutations in IRX5 impair craniofacial development and germ cell migration
via SDF1.

Bonnard C, Strobl AC, Shboul M, Lee H, Merriman B, Nelson SF,Ababneh OH, Uz E, Guran T, Kayserili H, Hamamy H, Reversade B.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Using homozygosity mapping and locus resequencing, we found that alterations in the homeodomain of the IRX5 transcription factor cause a recessive congenital disorder affecting face, brain, blood, heart,bone and gonad development. We found through in vivo modeling in Xenopus laevis embryos that Irx5 modulates the migration of progenitor cell populations in branchial arches and gonads by repressing Sdf1. We further found that transcriptional control by Irx5 is modulated by direct protein-protein interaction with two GATA zinc-finger proteins, GATA3 and TRPS1; disruptions of these proteins also cause craniofacial dysmorphisms. Our findings suggest that IRX proteins integrate combinatorial transcriptional inputs to regulate key signaling molecules involved in the ontogeny of multiple organs during embryogenesis and homeostasis.



Nature Genetics. 2011 Sep;41(9):1016-21.  

Goudie DRD'Alessandro MMerriman BLee HSzeverényi IAvery SO'Connor BDNelson SFCoats SEStewart A,Christie LPichert GFriedel JHayes IBurrows NWhittaker SGerdes AMBroesby-Olsen SFerguson-Smith MA,Verma CLunny DPReversade BLane EB.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Multiple self-healing squamous epithelioma (MSSE), also known as Ferguson-Smith disease (FSD), is an autosomal-dominant skin cancer condition characterized by multiple squamous-carcinoma-like locally invasive skin tumors that grow rapidly for a few weeks before spontaneously regressing, leaving scars. High-throughput genomic sequencing of a conservative estimate (24.2 Mb) of the disease locus on chromosome 9 using exon array capture identified independent mutations in TGFBR1 in three unrelated families. Subsequent dideoxy sequencing of TGFBR1 identified 11 distinct monoallelic mutations in 18 affected families, firmly establishing TGFBR1 as the causative gene. The nature of the sequence variants, which include mutations in the extracellular ligand-binding domain and a series of truncating mutations in the kinase domain, indicates a clear genotype-phenotype correlation between loss-of-function TGFBR1 mutations and MSSE. This distinguishes MSSE from the Marfan syndrome-related disorders in which missense mutations in TGFBR1 lead to developmental defects with vascular involvement but no reported predisposition to cancer.



Am J Hum Genet. 2010 Dec;10;87(6):768-78.

Tian J, Ling L, Shboul M, Lee H, O'Connor B, Merriman B, Nelson SF, Cool S, Ababneh OH, Al-Hadidy A, Masri A, Hamamy H, Reversade B.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

We delineated a syndromic recessive preaxial brachydactyly with partial duplication of proximal phalanges to 16.8 Mb over 4 chromosomes. High-throughput sequencing of all 177 candidate genes detected a truncating frameshift mutation in the gene CHSY1 encoding a chondroitin synthase with a Fringe domain. CHSY1 was secreted from patients' fibroblasts and was required for synthesis of chondroitin sulfate moieties. Noticeably, its absence triggered massive production of JAG1 and subsequent NOTCH activation, which could only be reversed with a wild-type but not a Fringe catalytically dead CHSY1 construct. In vitro, depletion of CHSY1 by RNAi knockdown resulted in enhanced osteogenesis in fetal osteoblasts and remarkable upregulation of JAG2 in glioblastoma cells. In vivo, chsy1 knockdown in zebrafish embryos partially phenocopied the human disorder; it increased NOTCH output and impaired skeletal, pectoral-fin, and retinal development. We conclude that CHSY1 is a secreted FRINGE enzyme required for adjustment of NOTCH signaling throughout human and fish embryogenesis and particularly during limb patterning.



Nature Genetics. 2009 Sep;41(9):1016-21.  

Mutations in PYCR1 cause cutis laxa with progeroid features.

Reversade B, Escande-Beillard N, Dimopoulou V, Fischer B, Chng S C, Li Y, Shboul M, et al.

Institute of Medical Biology, A*STAR, Singapore, Singapore.

Autosomal recessive cutis laxa (ARCL) describes a group of syndromal disorders that are often associated with a progeroid appearance, lax and wrinkled skin, osteopenia and mental retardation. Homozygosity mapping in several kindreds with ARCL identified a candidate region on chromosome 17q25. By high-throughput sequencing of the entire candidate region, we detected disease-causing mutations in the gene PYCR1. We found that the gene product, an enzyme involved in proline metabolism, localizes to mitochondria. Altered mitochondrial morphology, membrane potential and increased apoptosis rate upon oxidative stress were evident in fibroblasts from affected individuals. Knockdown of the orthologous genes in Xenopus and zebrafish led to epidermal hypoplasia and blistering that was accompanied by a massive increase of apoptosis. Our findings link mutations in PYCR1 to altered mitochondrial function and progeroid changes in connective tissues.



EMBO J. 2007 Jun 20;26(12):2955-65. 

The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning.

Sander V, Reversade B, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California-Los Angeles, 675 Charles Young Drive South, Los Angeles, CA 90095, USA.

We present a loss-of-function study using antisense morpholino (MO) reagents for the organizer-specific gene Goosecoid (Gsc) and the ventral genes Vent1 and Vent2. Unlike in the mouse Gsc is required in Xenopus for mesodermal patterning during gastrulation, causing phenotypes ranging from reduction of head structures-including cyclopia and holoprosencephaly-to expansion of ventral tissues in MO-injected embryos. The overexpression effects of Gsc mRNA require the expression of the BMP antagonist Chordin, a downstream target of Gsc. Combined Vent1 and Vent2 MOs strongly dorsalized the embryo. Unexpectedly, simultaneous depletion of all three genes led to a rescue of almost normal development in a variety of embryological assays. Thus, the phenotypic effects of depleting Gsc or Vent1/2 are caused by the transcriptional upregulation of their opposing counterparts. A principal function of Gsc and Vent1/2 homeobox genes might be to mediate a self-adjusting mechanism that restores the basic body plan when deviations from the norm occur, rather than generating individual cell types. The results may shed light on the molecular mechanisms of genetic redundancy.




Cell. 2006 Jan 13;124(1):147-59.

Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases.

Lee HX, Ambrosio AL, Reversade B, De Robertis EM.

Howard Hughes Medical Institute, Department of Biological Chemistry, University of California, Los Angeles, CA 90095, USA.

Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl -| Xlr -| Chd -| BMP --> P-Smad1 --> Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10(-8) M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism.
Commented in:



Cell. 2005 Dec 16;123(6):1147-60.

Regulation of ADMP and BMP2/4/7 at opposite embryonic  poles generates a self-regulating morphogenetic field.

Reversade B, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.  
Commented in:  
 Cell. 2005 Dec 16;123(6):982-4.

Bmp signaling: turning a half into a whole.

Kimelman D, Pyati UJ.

Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195, USA.

In a famous experiment over a century ago, Hans Spemann demonstrated that amphibians have a remarkable ability to compensate for perturbations to the embryo. In this issue of Cell, Reversade and De Robertis (2005) uncover the basis of this phenomenon. They demonstrate that interactions between bone morphogenetic proteins (Bmps) and their inhibitors on both the dorsal and ventral sides of the early Xenopus embryo are involved in creating the body plan.
Commented in:
 Development. 2005 Aug;132(15):3381-92.

Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive brain formation in Xenopus embryos.

Reversade B, Kuroda H, Lee H, Mays A, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA.

To address the patterning function of the Bmp2, Bmp4 and Bmp7 growth factors, we designed antisense morpholino oligomers (MO) that block their activity in Xenopus laevis. Bmp4 knockdown was sufficient to rescue the ventralizing effects caused by loss of Chordin activity. Double Bmp4 and Bmp7 knockdown inhibited tail development. Triple Bmp2/Bmp4/Bmp7 depletion further compromised trunk development but did not eliminate dorsoventral patterning. Unexpectedly, we found that blocking Spemann organizer formation by UV treatment or beta-Catenin depletion caused BMP inhibition to have much more potent effects, abolishing all ventral development and resulting in embryos having radial central nervous system (CNS) structures. Surprisingly, dorsal signaling molecules such as Chordin, Noggin, Xnr6 and Cerberus were not re-expressed in these embryos. We conclude that BMP inhibition is sufficient for neural induction in vivo, and that in the absence of ventral BMPs, Spemann organizer signals are not required for brain formation.


Genes Dev.
2005 May 1;19(9):1022-7.

Default neural induction: neuralization of dissociated Xenopus cells is mediated by Ras/MAPK activation.

Kuroda H, Fuentealba L, Ikeda A, Reversade B, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA.

Xenopus embryonic ectodermal cells dissociated for three or more hours differentiate into neural tissue instead of adopting their normal epidermal fate. This default type of neural induction occurs in the absence of Spemann's organizer signals and is thought to be caused by the dilution of endogenous BMPs into the culture medium. Unexpectedly, we observed that BMP ligands continue to signal in dissociated cells. Instead, cell dissociation induces a sustained activation of the Ras/MAPK pathway, which causes the phosphorylation of Smad1 at sites that inhibit the activity of this transcription factor. It is this activation of Ras/MAPK that is required for neuralization in dissociated ectoderm.

Development. 2005 May;132(10):2489-99.

Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm.

Zakin L, Reversade B, Kuroda H, Lyons KM, De Robertis EM.

Howard Hughes Medical Institute, and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA.

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.

 Development. 2003 Sep;130(17):4047-56.

The pro-BMP activity of Twisted gastrulation is independent of BMP binding.

Oelgeschlager M, Reversade B, Larrain J, Little S, Mullins MC, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California-Los Angeles, Los Angeles, CA 90095-1662, USA.

The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. We investigated the function of Tsg in embryonic patterning by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain were unable to bind BMP, yet ventralized the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity required an intact C-terminal domain and could block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity was specific for CR-containing proteins as it did not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants were stronger than the effect of Chordin loss-of-function in Xenopus or zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos.


Developmental Cell.
2003 Feb;4(2):219-30.

Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.

Oelgeschlager M, Kuroda H, Reversade B, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.

We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreased Chordin production. Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations. First, dorsalization by lithium chloride treatment was completely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely blocked the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation.



 Nat Cell Bio.  2002 Aug;4(8):599-604.

Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta.

Abreu JG, Ketpura NI, Reversade B, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Riverside, 9095-1662, USA.

Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-beta binding domains. Here we show that CTGF directly binds BMP4 and TGF-beta 1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-beta 1. These results show that CTGF inhibits BMP and activates TGF-beta signals by direct binding in the extracellular space.

 Development. 2001 Nov;128(22):4439-47.

Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling.

Larrain J, Oelgeschlager M, Ketpura NI, Reversade B, Zakin L, De Robertis EM.

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA.

Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). We have analyzed the molecular mechanism by which Tsg regulates BMP signaling. Overexpression of Tsg mRNA in Xenopus embryos has ventralizing effects similar to Xolloid, a metalloprotease that cleaves Chordin. In embryos dorsalized by LiCl treatment, microinjection of Xolloid or Tsg mRNA restores the formation of trunk-tail structures, indicating an increase in BMP signaling. Microinjection of Tsg mRNA leads to the degradation of endogenous Chordin fragments generated by Xolloid. The ventralizing activities of Tsg require an endogenous Xolloid-like activity, as they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay revealed that Tsg has two distinct and sequential activities on BMP signaling. First, Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. Second, after cleavage of Chordin by Xolloid, Tsg competes the residual anti-BMP activity of Chordin fragments and facilitates their degradation. This molecular pathway, in which Xolloid switches the activity of Tsg from a BMP antagonist to a pro-BMP signal once all endogenous full-length Chordin is degraded, may help explain how sharp borders between embryonic territories are generated.

 PNAS. 2000 Dec 19;97(26):14388-93.

Gene expression profiles in normal and Otx2-/- early gastrulating mouse embryos.

Zakin L, Reversade B, Virlon B, Rusniok C, Glaser P, Elalouf JM, Brulet P.

Unite d'Embyologie Moleculaire, Unite de Recherche Associee 1947, Centre National de la Recherche Scientifique, and Laboratoire de Genomique des Microorganismes Pathogenes, Institut Pasteur, 25 Rue du Docteur Roux, 75724, Paris Cedex 15, France.

The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and Otx2(-/-) 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation.