Kallikrein-8 mediates furin-independent Activin-A precursor processing to stimulate tumor growth in melanoma

M. Bulliard; K. Pinjusic; L. Iacobucci; C. Schmuziger; N. Fournier  et al.

Receptor binding of TGF-β and related ligands such as Activin-A requires cleavage of a furin site in their dimeric precursor proteins. Melanoma cells cleave one Activin-A subunit independently of furin and related proprotein convertases, raising questions of how this half-processed intermediate is generated and whether it influences tumor growth. Here, an siRNA library screen for proteases mediating this furin-independent “hemicleavage” identifies kallikrein (Klk)-8. While a KLK8 cleavage site in proActivin-A overlaps with the furin recognition sequence, its exposure is limited and requires prior transient acidification. Therefore, only furin efficiently converts proActivin-A to fully mature form both in tumor cells and in cell-free cleavage assays. Moreover, knockdown of Klk8 in syngeneic melanoma grafts suppresses Activin-A induced tumor growth, demonstrating that cleavage by only furin is not sufficient. Besides elucidating how Activin-A processing is regulated, our findings show that KLK8 holds promise as a target to mitigate Activin-A induced tumor growth.

DOI : 10.1038/s41467-025-57661-5

M⁶A methylation inhibits recruitment of the Dand5 3’UTR to the left-right determinant Bicc1

B. Rothé; M. Mendel; S. Fortier; D. B. Constam

In vertebrates, left-right (LR) asymmetry is specified by asymmetric decay of Dand5 messenger RNA (mRNA) mediated by the recruitment of the BicC family RNA binding protein 1 (Bicc1). Besides regulating organ laterality, Bicc1 is required to prevent cystic dilations in renal tubules and in pancreatic and bile ducts. However, validated target mRNAs are sparse in number, and how their binding to Bicc1 is regulated remains poorly understood. Bicc1 recruitment to Dand5 transcripts requires a conserved AGACGUGAC motif in the 3′UTR. Here, we report an N6-methyladenosine (m6A) in this sequence that disrupts binding to Bicc1 K homology (KH) domains in vitro, in stark contrast to IGF2BPs and FMR1, where m6A promotes RNA recognition by KH domains. We discuss the possible implications of this finding for LR axis formation and for a related role of Bicc1 in regulating specific target mRNAs in the kidney.

DOI : 10.1261/rna.080526.125

Stepwise release of Activin-A from its inhibitory prodomain is modulated by cysteines and requires furin coexpression to promote melanoma growth

K. Pinjusic; M. Bulliard; B. Rothé; S. Ansaryan; Y. C. Liu  et al.

The Activin-A precursor dimer can be cleaved by furin, but how this proteolytic maturation is regulated in vivo and how it facilitates access to signaling receptors is unclear. Here, analysis in a syngeneic melanoma grafting model shows that without furin coexpression, Activin-A failed to accelerate tumor growth, correlating with failure of one or both subunits to undergo cleavage in signal-sending cells, even though compensatory processing by host cells nonetheless sustained elevated circulating Activin-A levels. In reporter assays, furin-independent cleavage of one subunit enabled juxtacrine Activin-A signaling, whereas completion of proteolytic maturation by coexpressed furin or by recipient cells stimulated contact-independent activity, crosstalk with BMP receptors, and signal inhibition by follistatin. Mechanistically, Activin-A processing was modulated by allosteric disulfide bonds flanking the furin site. Disruption of these disulfide linkages with the prodomain enabled Activin-A binding to cognate type II receptors independently of proteolytic maturation. Stepwise proteolytic maturation is a novel mechanism to control Activin-A protein interactions and signaling.

DOI : 10.1038/s42003-024-07053-0

Tumor-educated Gr1+CD11b+cells drive breast cancer metastasis via OSM/IL-6/JAK-induced cancer cell plasticity

S. Peyvandi; M. Bulliard; A. Yilmaz; A. Kauzlaric; R. Marcone  et al.

Cancer cell plasticity contributes to therapy resistance and metastasis, which represent the main causes of cancer-related death, including in breast cancer. The tumor microenvironment drives cancer cell plasticity and metastasis, and unraveling the underlying cues may provide novel strategies for managing metastatic disease. Using breast cancer experimental models as a clinically relevant paracrine/autocrine axis instigating breast cancer cell plasticity and triggering metastasis.

DOI : 10.1172/JCI166847

Inhibition of anti-tumor immunity by melanoma cell-derived Activin-A depends on STING

K. Pinjusic; G. Ambrosini; J. Lourenco; N. Fournier; C. Iseli  et al.

The transforming growth factor-beta (TGF-beta) family member activin A (hereafter Activin-A) is overexpressed in many cancer types, often correlating with cancer-associated cachexia and poor prognosis. Activin-A secretion by melanoma cells indirectly impedes CD8+ T cell-mediated anti-tumor immunity and promotes resistance to immunotherapies, even though Activin-A can be proinflammatory in other contexts. To identify underlying mechanisms, we here analyzed the effect of Activin-A on syngeneic grafts of Braf mutant YUMM3.3 mouse melanoma cells and on their microenvironment using single-cell RNA sequencing. We found that the Activin-A-induced immune evasion was accompanied by a proinflammatory interferon signature across multiple cell types, and that the associated increase in tumor growth depended at least in part on pernicious STING activity within the melanoma cells. Besides corroborating a role for proinflammatory signals in facilitating immune evasion, our results suggest that STING holds considerable potential as a therapeutic target to mitigate tumor-promoting Activin-A signaling at least in melanoma.

DOI : 10.3389/fimmu.2023.1335207

Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3

B. Rothe; Y. Ikawa; Z. Zhang; T. A. Katoh; E. Kajikawa  et al.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.

DOI : 10.1371/journal.pbio.3002302

Antagonistic interactions among structured domains in the multivalent Bicc1-ANKS3-ANKS6 protein network govern phase transitioning of target mRNAs

B. Rothe; S. Fortier; C. Gagnieux; C. Schmuziger; D. B. Constam

The growing number of diseases linked to aberrant phase transitioning of ribonucleoproteins highlights the need to uncover how the interplay between multivalent protein and RNA interactions is regulated. Cytoplasmic granules of the RNA binding protein Bicaudal-C (Bicc1) are regulated by the ciliopathy proteins ankyrin (ANK) and sterile alpha motif (SAM) domain-containing ANKS3 and ANKS6, but whether and how target mRNAs are affected is unknown. Here, we show that head-to-tail polymers of Bicc1 nucleated by its SAM domain are interconnected by K homology (KH) domains in a protein meshwork that mediates liquid-to-gel transitioning of client transcripts. Moreover, while the dispersion of these granules by ANKS3 concomitantly released bound mRNAs, co-recruitment of ANKS6 by ANKS3 reinstated Bicc1 condensation and ribonucleoparticle assembly. RNA-independent Bicc1 polymerization and its dual regulation by ANKS3 and ANKS6 represent a new mechanism to couple the reversible immobilization of client mRNAs to controlled protein phase transitioning between distinct metastable states.

DOI : 10.1016/j.isci.2023.106855

Activin-A impairs CD8 T cell-mediated immunity and immune checkpoint therapy response in melanoma

K. Pinjusic; O. A. Dubey; O. Egorova; S. Nassiri; E. Meylan  et al.

Background Activin-A, a transforming growth factor beta family member, is secreted by many cancer types and is often associated with poor disease prognosis. Previous studies have shown that Activin-A expression can promote cancer progression and reduce the intratumoral frequency of cytotoxic T cells. However, the underlying mechanisms and the significance of Activin-A expression for cancer therapies are unclear. Methods We analyzed the expression of the Activin-A encoding gene INHBA in melanoma patients and the influence of its gain- or loss-of-function on the immune infiltration and growth of BRAF-driven YUMM3.3 and iBIP2 mouse melanoma grafts and in B16 models. Using antibody depletion strategies, we investigated the dependence of Activin-A tumor-promoting effect on different immune cells. Immune-regulatory effects of Activin-A were further characterized in vitro and by an adoptive transfer of T cells. Finally, we assessed INHBA expression in melanoma patients who received immune checkpoint therapy and tested whether it impairs the response in preclinical models. Results We show that Activin-A secretion by melanoma cells inhibits adaptive antitumor immunity irrespective of BRAF status by inhibiting CD8(+) T cell infiltration indirectly and even independently of CD4 T cells, at least in part by attenuating the production of CXCL9/10 by myeloid cells. In addition, we show that Activin-A/INHBA expression correlates with anti-PD1 therapy resistance in melanoma patients and impairs the response to dual anti-cytotoxic T-Lymphocyte associated protein 4/anti-PD1 treatment in preclinical models. Conclusions Our findings suggest that strategies interfering with Activin-A induced immune-regulation offer new therapeutic opportunities to overcome CD8 T cell exclusion and immunotherapy resistance.

DOI : 10.1136/jitc-2022-004533

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics

S. M. Leitao; B. Drake; K. Pinjusic; X. Pierrat; V. Navikas  et al.

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.

DOI : 10.1021/acsnano.1c05202

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

K. Minegishi; B. Rothe; K. R. Komatsu; H. Ono; Y. Ikawa  et al.

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3 untranslated region (3 ' -UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3 ' -UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3 ' -UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node. Questioning what regulates left-right asymmetry breaking in the mouse node: the authors identify a 200bp stretch of the Dand5 3'UTR where Bicc1 binds, and Cnot proteins downstream of calcium flow regulate the post-transcriptional regulation of Dand5 by Bicc1.

DOI : 10.1038/s41467-021-24295-2

The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity

B. Bragantini; C. Charron; M. Bourguet; A. Paul; D. Tiotiu  et al.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity. Biogenesis of small nucleolar RNAs ribonucleoproteins (snoRNPs) requires dedicated assembly machinery. Here, the authors show that a subset of snoRNP assembly factors interacts, genetically or directly, with factors modulating chromatin architecture, suggesting a link between ribosome formation and chromatin functions.

DOI : 10.1038/s41467-021-22077-4

Role of the RNA-binding protein Bicaudal-C1 and interacting factors in cystic kidney diseases

B. Rothe; C. Gagnieux; L. C. Leal-Esteban; D. B. Constam

Polycystic kidneys frequently associate with mutations in individual components of cilia, basal bodies or centriolar satellites that perturb complex protein networks. In this review, we focus on the RNA-binding protein Bicaudal-C1 (BICC1) which was found mutated in renal cystic dysplasia, and on its interactions with the ankyrin repeat and sterile a motif (SAM)-containing proteins ANKS3 and ANKS6 and associated kinases and their partially overlapping ciliopathy phenotypes. After reviewing BICC1 homologs in model organisms and their functions in mRNA and cell metabolism during development and in renal tubules, we discuss recent insights from cell-based assays and from structure analysis of the SAM domains, and how SAM domain oligomerization might influence multivalent higher order complexes that are implicated in ciliary signal transduction.

DOI : 10.1016/j.cellsig.2019.109499

The yeast C/D box snoRNA U14 adopts a "weak" K-turn like conformation recognized by the Snu13 core protein in solution

M.-E. Chagot; M. Quinternet; B. Rothe; B. Charpentier; J. Coutant  et al.

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3' side by a G.A/A.G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5' side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2'-O-methyl-transferase, Fibrillarin/Noplp. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The U.U and G.A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

DOI : 10.1016/j.biochi.2019.03.014

A novel cell-based sensor detecting the activity of individual basic proprotein convertases

K. Loew; K. Hardes; C. Fedeli; N. G. Seidah; D. B. Constam  et al.

The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides that may not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. Herein we report basic PC sensor (BPCS), a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. BPCS consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in the same membrane. Screening of selected candidate peptidomimetic inhibitors revealed that BPCS can readily distinguish between general and selective PC inhibitors in a high-throughput screening format. The robust and cost-effective assay format of BPCS makes it suitable to identify novel specific small-molecule inhibitors against basic PCs for therapeutic application. Its cell-based nature will allow screening for drug targets in addition to the catalytically active mature enzyme, including maturation, transport, and cellular factors that modulate the enzyme's activity. This broadened 'target range' will enhance the likelihood to identify novel small-molecule compounds that inhibit basic PCs in a direct or indirect manner and represents a conceptual advantage.

DOI : 10.1111/febs.14979

Comment on "Human dignity and gene editing"

D. B. Constam

DOI : 10.15252/embr.201847220

Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

B. Rothe; C. Leettola; L. Leal-Esteban; D. Cascio; S. Fortier  et al.

DOI : 10.1016/j.str.2017.12.002

Compartment-Specific Biosensors Reveal a Complementary Subcellular Distribution of Bioactive Furin and PC7

P. Ginefra; B. Filippi; P. Donovan; S. Bessonnard; D. Constam

DOI : 10.1016/j.celrep.2018.02.005

Role of Bicaudal C1 in renal gluconeogenesis and its novel interaction with the CTLH complex

L. Leal-Esteban; B. Rothe; S. Fortier; M. Isenschmid; D. Constam

DOI : 10.1371/journal.pgen.1007487

Paracrine Activin-A Signaling Promotes Melanoma Growth and Metastasis through Immune Evasion

P. Donovan; O. A. Dubey; S. Kallioinen; K. W. Rogers; K. Muehlethaler  et al.

The secreted growth factor Activin-A of the transforming growth factor beta family and its receptors can promote or inhibit several cancer hallmarks including tumor cell proliferation and differentiation, vascularization, lymphangiogenesis and inflammation. However, a role in immune evasion and its relationship with tumor-induced muscle wasting and tumor vascularization, and the relative contributions of autocrine versus paracrine Activin signaling remain to be evaluated. To address this, we compared the effects of truncated soluble Activin receptor IIB as a ligand trap, or constitutively active mutant type IB receptor versus secreted Activin-A or the related ligand Nodal in mouse and human melanoma cell lines and tumor grafts. We found that although cell-autonomous receptor activation arrested tumor cell proliferation, Activin-A secretion stimulated melanoma cell dedifferentiation and tumor vascularization by functional blood vessels, and it increased primary and metastatic tumor burden and muscle wasting. Importantly, in mice with impaired adaptive immunity, the tumor-promoting effect of Activin-A was lost despite sustained vascularization and cachexia, suggesting that Activin-A promotes melanoma progression by inhibiting antitumor immunity. Paracrine Activin-A signaling emerges as a potential target for personalized therapies, both to reduce cachexia and to enhance the efficacy of immunotherapies.

DOI : 10.1016/j.jid.2017.07.845

Epiblast-specific loss of HCF-1 leads to failure in anterior-posterior axis specification

S. Minocha; S. Bessonnard; T.-L. Sung; C. Moret; D. B. Constam  et al.

Mammalian Host-Cell Factor 1 (HCF-1), a transcriptional co-regulator, plays important roles during the cell-division cycle in cell culture, embryogenesis as well as adult tissue. In mice, HCF-1 is encoded by the X-chromosome-linked Hcfc1 gene. Induced Hcfc1(cKO/+) heterozygosity with a conditional knockout (cKO) allele in the epiblast of female embryos leads to a mixture of HCF-1-positive and -deficient cells owing to random X-chromosome inactivation. These embryos survive owing to the replacement of all HCF-1-deficient cells by HCF-1-positive cells during E5.5 to E8.5 of development. In contrast, complete epiblast-specific loss of HCF-1 in male embryos, Hcfc1(epiko/Y), leads to embryonic lethality. Here, we characterize this lethality. We show that male epiblast-specific loss of Hcfc1 leads to a developmental arrest at E6.5 with a rapid progressive cell-cycle exit and an associated failure of anterior visceral endoderm migration and primitive streak formation. Subsequently, gastrulation does not take place. We note that the pattern of Hcfc1(epiKO/y) lethality displays many similarities to loss of beta-catenin function. These results reveal essential new roles for HCF-1 in early embryonic cell proliferation and development. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.

DOI : 10.1016/j.ydbio.2016.08.008

Urine Fetuin-A is a biomarker of autosomal dominant polycystic kidney disease progression

N. Piazzon; F. Bernet; L. Guihard; W. N. Leonhard; S. Urfer  et al.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by numerous fluid-filled cysts that frequently result in end-stage renal disease. While promising treatment options are in advanced clinical development, early diagnosis and follow-up remain a major challenge. We therefore evaluated the diagnostic value of Fetuin-A as a new biomarker of ADPKD in human urine.

DOI : 10.1186/s12967-015-0463-7

PC7 and the related proteases Furin and Pace4 regulate E-cadherin function during blastocyst formation

S. Bessonnard; D. Mesnard; D. B. Constam

The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell-cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell-cell adhesion and fate allocation.

DOI : 10.1083/jcb.201503042

ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm

P. S. Ellis; S. Burbridge; S. Soubes; K. Ohyama; N. Ben-Haim  et al.

The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed Nodal. Exposure of prechordal mesoderm microcultures to Nodal-conditioned medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, targeted electroporation of Fgfr3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal-FGFR3 signalling and pSmad1/5/8, and show that proNodal-FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated.

DOI : 10.1242/dev.119628

Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA

B. Rothe; L. Leal-Esteban; F. Bernet; S. Urfer; N. Doerr  et al.

Loss of the RNA-binding protein Bicaudal-C (Bicc1) provokes renal and pancreatic cysts as well as ectopic Wnt/beta-catenin signaling during visceral left-right patterning. Renal cysts are linked to defective silencing of Bicc1 target mRNAs, including adenylate cyclase 6 (AC6). RNA binding of Bicc1 is mediated by N-terminal KH domains, whereas a C-terminal sterile alpha motif (SAM) self-polymerizes in vitro and localizes Bicc1 in cytoplasmic foci in vivo. To assess a role for multimerization in silencing, we conducted structure modeling and then mutated the SAM domain residues which in this model were predicted to polymerize Bicc1 in a left-handed helix. We show that a SAM-SAM interface concentrates Bicc1 in cytoplasmic clusters to specifically localize and silence bound mRNA. In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/beta-catenin pathway. Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects. We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms.

DOI : 10.1128/Mcb.00341-15

Bicaudal C1 promotes pancreatic NEUROG3+ endocrine progenitor differentiation and ductal morphogenesis

L. A. Lemaire; J. Goulley; Y. H. Kim; S. Carat; P. Jacquemin  et al.

In human, mutations in bicaudal C1 (BICC1), an RNA binding protein, have been identified in patients with kidney dysplasia. Deletion of Bicc1 in mouse leads to left-right asymmetry randomization and renal cysts. Here, we show that BICC1 is also expressed in both the pancreatic progenitor cells that line the ducts during development, and in the ducts after birth, but not in differentiated endocrine or acinar cells. Genetic inactivation of Bicc1 leads to ductal cell over-proliferation and cyst formation. Transcriptome comparison between WT and Bicc1 KO pancreata, before the phenotype onset, reveals that PKD2 functions downstream of BICC1 in preventing cyst formation in the pancreas. Moreover, the analysis highlights immune cell infiltration and stromal reaction developing early in the pancreas of Bicc1 knockout mice. In addition to these functions in duct morphogenesis, BICC1 regulates NEUROG3(+) endocrine progenitor production. Its deletion leads to a late but sustained endocrine progenitor decrease, resulting in a 50% reduction of endocrine cells. We show that BICC1 functions downstream of ONECUT1 in the pathway controlling both NEUROG3(+) endocrine cell production and ductal morphogenesis, and suggest a new candidate gene for syndromes associating kidney dysplasia with pancreatic disorders, including diabetes.

DOI : 10.1242/dev.114611

Pathogen-free husbandry conditions alleviate behavioral deficits and neurodegeneration in AD10 anti-NGF mice

S. Tzanoulinou; R. Brandi; I. Arisi; M. D'Onofrio; S. M. Urfer  et al.

It has been suggested that systemic infection, occurring during aging and chronic neurodegenerative diseases, can evoke an immune response that aggravates the progression of neurodegeneration and cognitive decline. It has been shown that the AD11 neurodegeneration mouse model, expressing a recombinant anti-nerve growth factor (NGF) antibody, shows a milder phenotype when housed in murine pathogen-free (MPF) conditions with respect to AD11 mice reared in conventional (CV) housing. AD10 mice, a variant of the anti-NGF AD11 model, expressing only an immunoglobulin light chain for the transgenic anti-NGF antibody, in the absence of the corresponding transgenic antibody chain VH, exhibit a complex neurodegenerative phenotype, similar to that of AD11 mice. Here we show that the AD10 transgenic mice, housed in murine pathogen-free conditions (MPF-AD10 mice), also display a milder behavioral and neurodegenerative phenotype compared to the corresponding mice kept under conventional housing conditions (CV-AD10). As a first step toward the identification of mechanisms underlying this difference, a differential gene expression profiling was performed on brains from CV-AD10 and MPF-AD10 mice, showing a decrease of the immune response and neuroinflammation gene expression in MPF-AD10 mice. Results suggest that the activation of the immune response gene expression in the CV-AD10, in a microbially unprotected environment, might contribute to a more severe and progressive neurodegenerative phenotype, compared to the MPF mice

DOI : 10.3233/JAD-131037

Nodal.Gdf1 Heterodimers with Bound Prodomains Enable Serum-independent Nodal Signaling and Endoderm Differentiation

C. Fuerer; M. C. Nostro; D. B. Constam

The TGF beta family member Nodal is central to control pluripotent stem cell fate, but its use as a stem cell differentiation factor is limited by low specific activity. During development, Nodal depends on growth and differentiation factor (Gdf)-1 and on the shared co-receptor Cryptic to specify visceral left-right axis asymmetry. We therefore asked whether the functionality of Nodal can be augmented by Gdf1. Because Nodal and Gdf1 coimmunoprecipitate each other, they were predicted to form heterodimers, possibly to facilitate diffusion or to increase the affinity for signaling receptors. Here, we report that Gdf1 suppresses an unexpected dependence of Nodal on serum proteins and that it is critically required for non-autonomous signaling in cells expressing Cryptic. Nodal, Gdf1, and their cleaved propeptides copurified as a heterodimeric low molecular weight complex that stimulated Activin receptor (Acvr) signaling far more potently than Nodal alone. Although heterodimerization with Gdf1 did not increase binding of Nodal to Fc fusions of co-receptors or Acvr extracellular domains, it was essential for soluble Acvr2 to inhibit Nodal signaling. This implies that Gdf1 potentiates Nodal activity by stabilizing a low molecular weight fraction that is susceptible to neutralization by soluble Acvr2. Finally, in differentiating human ES cells, endodermal markers were more efficiently induced by Nodal.Gdf1 than by Nodal, suggesting that Nodal.Gdf1 is an attractive new reagent to direct stem cell differentiation.

DOI : 10.1074/jbc.M114.550301

Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice

W. C. Wetsel; R. M. Rodriguiz; J. Guillemot; E. Rousselet; R. Essalmani  et al.

PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.

DOI : 10.1073/pnas.1314698110

De novo DNA methylation of endogenous retroviruses is shaped by KRAB-ZFPs/KAP1 and ESET

H. M. Rowe; M. Friedli; S. Offner; S. Verp; D. Mesnard  et al.

Endogenous retroviruses (ERVs) undergo de novo DNA methylation during the first few days of mammalian embryogenesis, although the factors that control the targeting of this process are largely unknown. We asked whether KAP1 (KRAB-associated protein 1) is involved in this mechanism because of its previously defined role in maintaining the silencing of ERVs through the histone methyltransferase ESET and histone H3 lysine 9 trimethylation. Here, we demonstrate that introduced ERV sequences are sufficient to direct rapid de novo methylation of a flanked promoter in embryonic stem (ES) cells. This mechanism requires the presence of an ERV sequence-recognizing KRAB zinc-finger protein (ZFP) and both KAP1 and ESET. Furthermore, this process can also take place on a strong cellular promoter and leads to methylation signatures that are subsequently maintained in vivo throughout embryogenesis. Finally, we show that methylation of ERVs residing in the genome is affected by knockout of KAP1 in early embryos. KRAB-ZFPs, KAP1 and ESET are thus likely to be responsible for the early embryonic instatement of stable epigenetic marks at ERV-containing loci.

DOI : 10.1242/dev.087585

Furin Is the Primary in Vivo Convertase of Angiopoietin-like 3 and Endothelial Lipase in Hepatocytes

R. Essalmani; D. Susan-Resiga; A. Chamberland; M.-C. Asselin; M. Canuel  et al.

The proprotein convertases (PCs) furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knockout (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because furin and PC5/6 complete KOs are lethal, we used mice lacking furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by furin only, and extracellularly mainly by PACE4. In vivo, the absence of furin in hepatocytes reduced by similar to 50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by furin. However, the lack of furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of furin in liver would not be expected to modify the plasma lipid profiles.

DOI : 10.1074/jbc.M113.501304

Bicc1 links the regulation of cAMP signaling in polycystic kidneys to microRNA-induced gene silencing

N. Piazzon; C. Maisonneuve; I. Guilleret; S. Rotman; D. B. Constam

Genetic defects in autosomal dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are upregulated in cystic kidneys of Bicc1-/- knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. Repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules

DOI : 10.1093/jmcb/mjs027

Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells Is Revealed by the ShcD/RaLP Adaptor Protein

M. Y. Turco; L. Furia; A. Dietze; L. F. Diaz; S. Ronzoni  et al.

The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naive (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity. STEM CELLS2012;30:24232436

DOI : 10.1002/stem.1217

Automated design of ligands to polypharmacological profiles

J. Besnard; G. F. Ruda; V. Setola; K. Abecassis; R. M. Rodriguiz  et al.

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.

DOI : 10.1038/nature11691

The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities

D. Mesnard; M. Donnison; C. Fuerer; P. L. Pfeffer; D. B. Constam

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

DOI : 10.1101/gad.16738711

Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

D. Susan-Resiga; R. Essalmani; J. Hamelin; M.-C. Asselin; S. Benjannet  et al.

DOI : 10.1074/jbc.M111.233577

Two mutations in human BICC1 resulting in Wnt pathway hyperactivity associated with cystic renal dysplasia

M. R.-C. Kraus; S. Clauin; Y. Pfister; M. Di Maïo; T. Ulinski  et al.

Bicaudal C homologue 1 (Bicc1) knockout in mice causes polycystic kidney disease and pancreas development defects, including a reduction in insulin-producing β-cells and ensuing diabetes. We therefore screened 137 patients with renal abnormalities or association of early-onset diabetes and renal disease for genetic alterations in BICC1. We identified two heterozygous mutations, one nonsense in the first K Homology (KH) domain and one missense in the sterile alpha motif (SAM) domain. In mice, Bicc1 blocks canonical Wnt signaling, mostly via its SAM domain. We show that the human BICC1, similar to its mouse counterpart, blocks canonical Wnt signaling. The nonsense mutation identified results in a complete loss of Wnt inhibitory activity. The point mutation in the SAM domain has a similar effect to a complete SAM domain deletion, resulting in a 22% loss of activity. Hum Mutat 33:86-90, 2012. © 2011 Wiley Periodicals, Inc.

DOI : 10.1002/humu.21610

Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

D. Mesnard; D. B. Constam

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β-related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface-targeted fluorescent biosensor (cell surface-linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

DOI : 10.1083/jcb.201005026

KAP1 controls endogenous retroviruses in embryonic stem cells

H. M. Rowe; J. Jakobsson; D. Mesnard; J. Rougemont; S. Reynard  et al.

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

DOI : 10.1038/nature08674

Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow

C. Maisonneuve; I. Guilleret; P. Vick; T. Weber; P. Andre  et al.

Polycystic diseases and left-right (LR) axis malformations are frequently linked to cilia defects. Renal cysts also arise in mice and frogs lacking Bicaudal C (BicC), a conserved RNA-binding protein containing K-homology (KH) domains and a sterile alpha motif (SAM). However, a role for BicC in cilia function has not been demonstrated. Here, we report that targeted inactivation of BicC randomizes left-right (LR) asymmetry by disrupting the planar alignment of motile cilia required for cilia-driven fluid flow. Furthermore, depending on its SAM domain, BicC can uncouple Dvl2 signaling from the canonical Wnt pathway, which has been implicated in antagonizing planar cell polarity (PCP). The SAM domain concentrates BicC in cytoplasmic structures harboring RNA-processing bodies (P-bodies) and Dvl2. These results suggest a model whereby BicC links the orientation of cilia with PCP, possibly by regulating RNA silencing in P-bodies.

DOI : 10.1242/dev.038174

Cripto recruits Furin and PACE4 and controls Nodal trafficking during proteolytic maturation

M. Blanchet; J. Le Good; D. Mesnard; V. Oorschot; S. Baflast  et al.

The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP-Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.

DOI : 10.1038/emboj.2008.174

Cripto localizes nodal at the limiting membrane of early endosomes

M. Blanchet; J. Le Good; V. Oorschot; S. Baflast; G. Minchiotti  et al.

Cripto is a glycosylphosphatidylinositol (GPI)-anchored co-receptor of Nodal and several other transforming growth factor-beta (TGF-beta) family ligands. It contains an epidermal growth factor (EGF)-like motif and a Cripto-FRL1-Cryptic (CFC) domain, which are conserved in a family of EGF-CFC proteins. The EGF domain is thought to recruit Nodal, whereas the CFC domain mediates binding to activin receptor-like kinase 4 (ALK4). We found that the EGF-like motif of Cripto was not essential for its binding to Nodal. However, through residues phenylalanine 78 and glycine 71, Cripto enriched Nodal at the limiting membrane of early endosomes. Similarly, residues in the CFC domain that mediate binding of Cripto to ALK4 were required to attenuate sequestration of Nodal in the endosomal lumen. Thus, we propose that Cripto stimulates Nodal activity by localizing it at the interface of endosomes with cytoplasmic effectors. To our knowledge, Cripto is the first GPI-anchored protein shown to control intraendosomal sorting of its associated cargo.

DOI : 10.1126/scisignal.1165027

VACTERL/caudal regression/Currarino syndrome-like malformations in mice with mutation in the proprotein convertase Pcsk5

D. Szumska; G. Pieles; R. Essalmani; M. Bilski; D. Mesnard  et al.

We have identified an ethylnitrosourea (ENU)-induced recessive mouse mutation (Vcc) with a pleiotropic phenotype that includes cardiac, tracheoesophageal, anorectal, anteroposterior patterning defects, exomphalos, hindlimb hypoplasia, a presacral mass, renal and palatal agenesis, and pulmonary hypoplasia. It results from a C470R mutation in the proprotein convertase PCSK5 (PC5/6). Compound mutants (Pcsk5(Vcc/null)) completely recapitulate the Pcsk5(Vcc/Vcc) phenotype, as does an epiblast-specific conditional deletion of Pcsk5. The C470R mutation ablates a disulfide bond in the P domain, and blocks export from the endoplasmic reticulum and proprotein convertase activity. We show that GDF11 is cleaved and activated by PCSK5A, but not by PCSK5A-C470R, and that Gdf11-deficient embryos, in addition to having anteroposterior patterning defects and renal and palatal agenesis, also have a presacral mass, anorectal malformation, and exomphalos. Pcsk5 mutation results in abnormal expression of several paralogous Hox genes (Hoxa, Hoxc, and Hoxd), and of Mnx1 (Hlxb9). These include known Gdf11 targets, and are necessary for caudal embryo development. We identified nonsynonymous mutations in PCSK5 in patients with VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb malformation OMIM 192350) and caudal regression syndrome, the phenotypic features of which resemble the mouse mutation. We propose that Pcsk5, at least in part via GDF11, coordinately regulates caudal Hox paralogs, to control anteroposterior patterning, nephrogenesis, skeletal, and anorectal development

DOI : 10.1101/gad.479408

Cripto promotes A-P axis specification independently of its stimulatory effect on Nodal autoinduction

D. D'Andrea; G. L. Liguori; J. A. Le Good; E. Lonardo; O. Andersson  et al.

The EGF-CFC gene cripto governs anterior-posterior (A-P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII-activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal-ALK4-Smad2 signaling both in embryonic stem cells and cell-based assays. In cripto(F78A/F78A) mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto-null mutants, cripto(F78A/F78A) embryos establish an A-P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal-Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A-P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal-ALK4-Smad2 signaling pathway

DOI : 10.1083/jcb.200709090

The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4

N. Ben-Haim; C. Lu; M. Guzman-Ayala; L. Pescatore; D. Mesnard  et al.

During early mouse development, the subtilisin-like proprotein convertases (SPC) Furin and PACE4 pattern the primitive ectoderm and visceral endoderm, presumably by activating the TGFss-related Nodal precursor. Here, mutation of the SPC motif provides direct evidence that Nodal processing is essential to specify anterior visceral endoderm and mesendoderm. Surprisingly, however, the Nodal precursor binds and activates activin receptors to maintain expression of Furin, PACE4, and Bmp4 in extraembryonic ectoderm at a distance from the Nodal source. In return, Bmp4 induces Wnt3, which amplifies Nodal expression in the epiblast and mediates induction of mesoderm. We conclude that uncleaved Nodal sustains the extraembryonic source of proprotein convertases and Bmp4 to amplify Nodal signaling in two nonredundant feedback loops with dual timescales and to localize primitive streak formation at the posterior pole. Based on mathematical modeling, we discuss how these sequential loops control cell fate.

DOI : 10.1016/j.devcel.2006.07.005

Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning

D. Mesnard; M. Guzman-Ayala; D. B. Constam

Anteroposterior (AP) polarity in the mammalian embryo is specified during gastrulation when naive progenitor cells in the primitive ectoderm are recruited into the primitive streak to form mesoderm and endoderm. At the opposite pole, this process is inhibited by signals previously induced in distal visceral endoderm (DVE). Both DVE and primitive streak formation, and hence positioning of the AP axis, rely on the TGFbeta family member Nodal and its proprotein convertases Furin and Pace4. Here, we show that Nodal and Furin are initially co-expressed in the primitive endoderm together with a subset of DVE markers such as Lefty1 and Hex. However, with the appearance of extra-embryonic ectoderm (ExE), DVE formation is transiently inhibited. During this stage, Nodal activity is essential to specify embryonic VE and restrict the expression of Furin to the extra-embryonic region. Activation of Nodal is also necessary to maintain determinants of pluripotency such as Oct4, Nanog and Foxd3 during implantation, and to stimulate elongation of the egg cylinder, before inducing DVE and germ layer formation. We conclude that Nodal is already activated in primitive endoderm, but induces a functional DVE only after promoting the expansion of embryonic VE and pluripotent progenitor cells in the epiblast.

DOI : 10.1242/dev.02413

Nodal stability determines signaling range

J. A. Le Good; K. Joubin; A. J. Giraldez; N. Ben-Haim; S. Beck  et al.

Secreted TGFbeta proteins of the Nodal family pattern the vertebrate body axes and induce mesoderm and endoderm . Nodal proteins can act as morphogens , but the mechanisms regulating their activity and signaling range are poorly understood. In particular, it has been unclear how inefficient processing or rapid turnover of the Nodal protein influences autocrine and paracrine signaling properties . Here, we evaluate the role of Nodal processing and stability in tissue culture and zebrafish embryos. Removal of the pro domain potentiates autocrine signaling but reduces Nodal stability and signaling range. Insertion of an N-glycosylation site present in several related TGFbeta proteins increases the stability of mature Nodal. The stabilized form of Nodal acts at a longer range than the wild-type form. These results suggest that increased proteolytic maturation of Nodal potentiates autocrine signaling, whereas increased Nodal stability extends paracrine signaling.

DOI : 10.1016/j.cub.2004.12.062

Asymmetric Nodal expression in the mouse is governed by the combinatorial activities of two distinct regulatory elements

S. D. Vincent; D. P. Norris; J. A. Le Good; D. B. Constam; E. J. Robertson

In all vertebrates, invariant left/right (L/R) positioning and organization of the internal viscera is controlled by a conserved pathway. Nodal, a member of the TGFbeta superfamily is a critical upstream component responsible for initiating L/R axis determination. Asymmetric Nodal expression in the node preceeds and foreshadows morphological L/R asymmetry. Here we address the mechanism of Nodal activation in the left LPM by studying the function of a novel enhancer element, the AIE. We show this element is exclusively active in cells of the left lateral plate mesoderm (LPM) and is not itself responding to Nodal asymmetry. To test the hypothesis that this element may initiate asymmetric Nodal expression in the LPM, we deleted it from the mouse germ line. Mice homozygous for the AIE deletion (Nodal(deltaaie/deltaaie)) show no defects. However, we find that the AIE contributes to regulating the level of asymmetric Nodal activity; analysis of transheterozygous embryos (Nodal(deltaaie/null)) shows reduced Nodal expression in the left LPM associated with a low penetrance of L/R defects. Our findings point to the existence of two independent pathways that control Nodal expression in the left LPM.

DOI : 10.1016/j.mod.2004.06.002

Nodal protein processing and fibroblast growth factor 4 synergize to maintain a trophoblast stem cell microenvironment

M. Guzman-Ayala; N. Ben-Haim; S. Beck; D. B. Constam

Before implantation in the uterus, mammalian embryos set aside trophoblast stem cells that are maintained in the extraembryonic ectoderm (ExE) during gastrulation to generate the fetal portion of the placenta. Their proliferation depends on diffusible signals from neighboring cells in the epiblast, including fibroblast growth factor 4 (Fgf4). Here, we show that Fgf4 expression is induced by the transforming growth factor beta-related protein Nodal. Together with Fgf4, Nodal also acts directly on neighboring ExE to sustain a microenvironment that inhibits precocious differentiation of trophoblast stem cells. Because the ExE itself produces the proteases Furin and PACE4 to activate Nodal, it represents the first example, to our knowledge, of a stem cell compartment that actively maintains its own microenvironment.

DOI : 10.1073/pnas.0405429101

Extraembryonic proteases regulate Nodal signalling during gastrulation

S. Beck; J. A. Le Good; M. Guzman; N. Ben Haim; K. Roy  et al.

During gastrulation, a cascade of inductive tissue interactions converts pre-existing polarity in the mammalian embryo into antero-posterior pattern. This process is triggered by Nodal, a protein related to transforming growth factor-beta (TFG-beta) that is expressed in the epiblast and visceral endoderm, and its co-receptor Cripto, which is induced downstream of Nodal. Here we show that the proprotein convertases Spc1 and Spc4 (also known as Furin and Pace4, respectively) are expressed in adjacent extraembryonic ectoderm. They stimulate Nodal maturation after its secretion and are required in vivo for Nodal signalling. Embryo explants deprived of extraembryonic ectoderm phenocopy Spc1(-/-); Spc4(-/-) double mutants in that endogenous Nodal fails to induce Cripto. But recombinant mature Nodal, unlike uncleaved precursor, can efficiently rescue Cripto expression. Cripto is also expressed in explants treated with bone morphogenetic protein 4 (BMP4). This indicates that Nodal may induce Cripto through both a signalling pathway in the embryo and induction of Bmp4 in the extraembryonic ectoderm. A lack of Spc1 and Spc4 affects both pathways because these proteases also stimulate induction of Bmp4.

DOI : 10.1038/ncb890

Tissue-specific requirements for the proprotein convertase furin/SPC1 during embryonic turning and heart looping

D. B. Constam; E. J. Robertson

Furin, the mammalian prototype of a family of serine proteases, is required for ventral closure and axial rotation, and formation of the yolk sac vasculature. Here we show additionally that left-sided expression of pitx2 and lefty-2 are also perturbed in Furin-deficient embryos. These tissue abnormalities are preceded by a marked delay in the expansion of the definitive endoderm during gastrulation. Using a chimera approach, we show that Furin activity is required in epiblast derivatives, including the primitive heart, gut and extraembryonic mesoderm, whereas it is nonessential in the visceral endoderm. Thus, chimeric embryos, derived by injecting wild-type embryonic stem (ES) cells into fur(-/-) blastocysts, develop normally until at least 9.5 d.p.c. In contrast, Furin-deficient chimeras developing in the context of wild-type visceral endoderm fail to undergo ventral closure, axial rotation and yolk sac vascularization. Fur(-/-) cells are recruited into all tissues examined, including the yolk sac vasculature and the midgut, even though these structures fail to form in fur mutants. The presence of wild-type cells in the gut strikingly correlates with the ability of chimeric embryos to undergo turning. Overall, we conclude that Furin activity is essential in both extraembryonic and precardiac mesoderm, and in definitive endoderm derivatives.

SPC4/PACE4 regulates a TGFbeta signaling network during axis formation

D. B. Constam; E. J. Robertson

In vertebrates, specification of anteroposterior (A/P) and left-right (L/R) axes depends on TGFbeta-related signals, including Nodal, Lefty, and BMPs. Endoproteolytic maturation of these proteins is probably mediated by the proprotein convertase SPC1/Furin. In addition, precursor processing may be regulated by related activities such as SPC4 (also known as PACE4). Here, we show that a proportion of embryos lacking SPC4 develop situs ambiguus combined with left pulmonary isomerism or complex craniofacial malformations including cyclopia, or both. Gene expression analysis during early somite stages indicates that spc4 is genetically upstream of nodal, pitx2, lefty1, and lefty2 and perhaps maintains the balance between Nodal and BMP signaling in the lateral plate that is critical for L/R axis formation. Furthermore, genetic interactions between nodal and spc4, together with our analysis of chimeric embryos, strongly suggest that during A/P axis formation, SPC4 acts primarily in the foregut. These findings establish an important role for SPC4 in patterning the early mouse embryo.

Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases

D. B. Constam; E. J. Robertson

Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Here we show that processing of Nodal and Myc-tagged BMP4 is significantly enhanced by SPC1/Furin or SPC4/PACE4, providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions.

DOI : 10.1083/jcb.144.1.139

Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin

A. J. Roebroek; L. Umans; I. G. Pauli; E. J. Robertson; F. van Leuven  et al.

We have examined the role of Furin in postimplantation-stage mouse embryos by analyzing both the expression pattern of fur mRNA and the developmental consequences of a loss-of-function mutation at the fur locus. At early stages (day 7.5), fur mRNA is abundant in extraembryonic endoderm and mesoderm, anterior visceral endoderm, and in precardiac mesoderm. 1 day later fur is expressed throughout the heart tube and in the lateral plate mesoderm, notochordal plate and definitive gut endoderm. Embryos lacking Furin die between days 10.5 and 11.5, presumably due to hemodynamic insufficiency associated with severe ventral closure defects and the failure of the heart tube to fuse and undergo looping morphogenesis. Morphogenesis of the yolk sac vasculature is also abnormal, although blood islands and endothelial precursors form. Analysis of cardiac and endodermal marker genes shows that while both myocardial precursors and definitive endoderm cells are specified, their numbers and migratory properties are compromised. Notably, mutant embryos fail to undergo axial rotation, even though Nodal and eHand, two molecular markers of left-right asymmetry, are appropriately expressed. Overall, the present data identify Furin as an important activator of signals responsible for ventral closure and embryonic turning.

Cloning of the human puromycin-sensitive aminopeptidase and evidence for expression in neurons

A. R. Tobler; D. B. Constam; A. Schmitt-Gräff; U. Malipiero; R. Schlapbach  et al.

The puromycin-sensitive aminopeptidase (PSA) is thought to contribute to the degradation of enkephalins. Besides being the most abundant aminopeptidase in the brain, PSA is expressed in other organs as well. From a human fetal brain cDNA library, we have isolated a cDNA encoding the human PSA (huPSA) protein. The isolated cDNA gave rise to a protein with a molecular mass of 99 kDa. Compared with mouse PSA, homology at the amino acid and cDNA level was 98 and 93%, respectively. Translation of the huPSA was found to be initiated at the second of two possible start codons, as shown by studies with antibodies directed against peptide sequences of both potential N-terminal regions. Northern blot analysis with RNA isolated from different human organs demonstrated that the huPSA transcript is strongest but not exclusively expressed in the brain. Vesicular stomatitis virus epitope-tagged huPSA protein was expressed in HeLa cells and found to be localized in the cytoplasm, especially in the perinuclear region. By in situ hybridization, huPSA transcript could be identified in cortical and cerebellar neurons, whereas glial cells and blood vessels remained negative.

DOI : 10.1046/j.1471-4159.1997.68030889.x

SPC4, SPC6, and the novel protease SPC7 are coexpressed with bone morphogenetic proteins at distinct sites during embryogenesis

D. B. Constam; M. Calfon; E. J. Robertson

In the present study, we screened for subtilisin-like proprotein convertases (SPCs) that potentially regulate the activation of known growth factors during embryonic development. We isolated a novel protease, SPC7, as well as several known SPCs. SPC7, like SPC1, is expressed ubiquitously throughout development. In contrast, SPC4 and SPC6 exhibit dynamic expression patterns. SPC4 transcripts were initially detected in the granulosa cells of secondary follicles. Shortly after implantation, SPC4 transcripts are localized to extraembryonic cell populations, and at later stages are detected in discrete tissues including the primitive gut, heart, neural tube, and limb buds. Within the limb buds, SPC4 mRNA is most abundant in the apical ectodermal ridge (AER). At later stages of limb development, SPC4 mRNA is strongly expressed in cartilage and in the interdigital mesenchyme. In contrast, high SPC6 mRNA levels are detected in somites, the dorsal surface ectoderm, and in vertebral cartilage primordia. In limb buds, SPC6 is strongly expressed in the AER, and at later stages in dorsal mesenchyme. A comparison of these expression patterns with those of several bone morphogenetic proteins (BMPs) indicates that processing of these growth factors may be limited by the local availability of SPCs.

DOI : 10.1083/jcb.134.1.181