Manuscripts (pdf) & Abstracts


2014


J Invest Dermatol. 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.


2013


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

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.



Hum Genet. 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.


2012 


Mol Biol Cell. 2012 Dec;23(24):4713-24. 

Jodoin JN, Shboul M, Sitaram P, Zein-Sabatto H, Reversade B, Lee E, Lee LA.

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


Recruitment of dynein motors to the nuclear surface is an essential step for nucleus-centrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2-Bicaudal D2 (BICD2) and Nup133- centromere protein F (CENP-F) networks. We previously reported that the asunder (asun) gene is required in Drosophila spermatocytes for perinuclear dynein localization and nucleus-centrosome coupling at G2/M of male meiosis. We show here that male germline expression of mammalian Asunder (ASUN) protein rescues asun flies, demonstrating evolutionary conservation of function. In cultured human cells, we find that ASUN down-regulation causes reduction of perinuclear dynein in prophase of mitosis. Additional defects after loss of ASUN include nucleus-centrosome uncoupling, abnormal spindles, and multinucleation. Coimmunoprecipitation and overlapping localization patterns of ASUN and lissencephaly 1 (LIS1), a dynein adaptor, suggest that ASUN interacts with dynein in the cytoplasm via LIS1. Our data indicate that ASUN controls dynein localization via a mechanism distinct from that of either BICD2 or CENP-F. We present a model in which ASUN promotes perinuclear enrichment of dynein at G2/M that facilitates BICD2- and CENP-F-mediated anchoring of dynein to nuclear pore complexes.


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.



2011

 

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

Multiple self-healing squamous epithelioma is caused by a disease-specific spectrum of mutations in TGFBR1.


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.



2010

 

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.



2009

 

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.

 

2007


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.

 

2006

 

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:

 


2005


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. kimelman@u.washington.edu

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.
 
 
2003

 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.

 

2002

 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.


2001 
 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.




 
2000
 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.
 
 
1997
 Biochem Cell Biol. 1997;75(5):623-32.

Characterization of a cDNA encoding a novel band 4.1-like protein in zebrafish.

Kelly GM, Reversade B.

Department of Zoology, University of Western Ontario, London, Canada. gkelly@julian.uwo.ca

Membrane skeleton protein 4.1 and other members of a family of proteins that link the cytoskeleton to the plasma membrane may play an integral role in cell communication during development. The polymerase chain reaction and degenerate oligodeoxynucleotide primers to consensus sequences in the putative membrane-binding domain of the protein 4.1 superfamily were used to isolate cDNAs encoding members of the zebrafish protein 4.1 family. Zebrafish stage- and tissue-specific first strand cDNA was used in the PCR. After the reaction, amplicons of the predicted size were sequenced to confirm their relationship to the protein 4.1 superfamily. One cDNA, with a high degree of similarity to a mouse novel band 4.1-like cDNA, was used to probe a zebrafish adult brain library. A 2.4-kb cDNA was isolated and found to encode a 619 amino acid polypeptide homologous to mouse novel band 4.1-like protein 4. Zebrafish nbl4 mRNA is maternally supplied and is expressed throughout embryogenesis. In adults, nbl4 is found in the ovary, eye, heart, and brain, but not in gut or skeletal muscle. When synthetic nbl4 mRNA is translated in vitro it binds calmodulin in a calcium-dependent manner. These data indicate that zebrafish nbl4 is a maternal transcript owing to its presence before the midblastula transition, and it is present later on in specific adult structures. The ability to bind calmodulin would suggest that the function of nbl4 protein may be potentially regulated via a calcium-calmodulin dependent mechanism.