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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 200 bp 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.

SUMMARYPerturbations in biomolecular condensates that form by phase-transitioning are linked to a growing number of degenerative diseases. For example, mutations in a multivalent interaction network of the Ankyrin (ANK) and sterile alpha motif (SAM) domain-containing ANKS3 and ANKS6 proteins with the RNA-binding protein Bicaudal-C1 (Bicc1) associate with laterality defects and chronic kidney diseases known as ciliopathies. However, insights into the mechanisms that control RNA condensation in ribonucleoprotein particles (RNPs) are scarce. Here, we asked whether heterooligomerization modulates Bicc1 binding to RNA. Reconstitution assays in vitro and live imaging in vivo show that a K homology (KH) repeat of Bicc1 self-interacts and synergizes with SAM domain self-polymerization independently of RNA to concentrate bound mRNAs in gel-like granules that can split or fuse with each other. Importantly, emulsification of Bicc1 by ANKS3 inhibited binding to target mRNAs, whereas condensation by ANKS6 co-recruitment increased it by liberating the KH domains from ANKS3. Our findings suggest that the perturbation of Bicc1-Anks3-Anks6 RNP dynamics is a likely cause of associated ciliopathies.

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 microscopy1, EM2, AFM3). However, time-resolved imaging techniques tend to either have 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. By utilizing recent advances in nanopositioning4, nanopore fabrication5, microelectronics6, and controls engineering7 we developed a microscopy method that can resolve spatiotemporally diverse three-dimenional processes on the cell membrane at sub-5nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of sub-second to days, imagining 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.

SUMMARYMolecular 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 3’ untranslated region (3’-UTR) of Dand5 mRNA is necessary and sufficient for the left-sided decay and is responsive to the flow direction, loss of the cation channel Pkd2, and Ca2+. The 200-nucleotide proximal-most portion of the 3’-UTR, which is conserved among mammals, is essential for the asymmetric mRNA decay and binds Bicc1, an RNA binding protein specifically expressed at the node. Bicc1 preferentially recognizes GAC and GACR sequences in RNA, and these motifs are enriched in the 200-nucleotide region of the 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. Our results thus suggest that leftward fluid flow induces binding of Bicc1 to the 3’-UTR of Dand5 mRNA in crown cells on the left side of the node, and that consequent recruitment of Ccr4-Not mediates mRNA degradation.

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 α 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.

EMBO Reports (2019) 20: e47220[OpenUrl][1][FREE Full Text][2] In EMBO Reports 19(10): e46789 [1], de Miguel Beriain argues that “[u]sing human dignity as an argument against modifying the human genome and germline is a logical fallacy”. He maintains that logical considerations must lead to the conclusion that “… respect for human dignity should actually support arguments to continue with genetic editing of embryos”. The topic is of great urgency: The biomedical community, policymakers, and the public struggle to come to a consensus as to whether and under which conditions human genome editing should be admissible or desirable. As a concerned citizen and researcher in the life sciences, I would like to discuss why I find the author's arguments unconvincing and the conclusions misleading. If one believes that our dignity can only remain undiminished if the human genome remains unchanged, we cannot tolerate any (even natural) new mutations. This argument rests on the author's assumption that “unchanged” can and should be defined as “never mutating”. However, human dignity is not violated by the fact that human DNA can and does change. Human dignity suffers violation when some individuals are invested with authority to engineer the genomes of others who are not yet born. In other words, the relevant question is not how heritable information changes, but rather who is entitled to change it. The belief that only nature or a creator should have … [1]: {openurl}?query=rft.jtitle%253DEMBO%2BReports%26rft_id%253Dinfo%253Adoi%252F10.15252%252Fembr.201847220%26rft_id%253Dinfo%253Apmid%252F30498078%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [2]: /lookup/ijlink?linkType=FULL&journalCode=embor&resid=20/1/e47220&atom=%2Fembor%2F20%2F1%2Fe47220.atom

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.

Altered glucose and lipid metabolism fuel cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Renal cysts also associate with mutations in Bicaudal C1 (Bicc1) or in its self-polymerizing sterile alpha motif (SAM). Here, we found that Bicc1 maintains normoglycemia and the expression of the gluconeogenic enzymes FBP1 and PEPCK in kidneys. A proteomic screen revealed that Bicc1 interacts with the C-Terminal to Lis-Homology domain (CTLH) complex. Since the orthologous Gid complex in S. cerevisae targets FBP1 and PEPCK for degradation, we mapped the topology among CTLH subunits and found that SAM-mediated binding controls Bicc1 protein levels, whereas Bicc1 inhibited the accumulation of several CTLH subunits. Under the conditions analyzed, Bicc1 increased FBP1 protein levels independently of the CTLH complex. Besides linking Bicc1 to cell metabolism, our findings reveal new layers of complexity in the regulation of renal gluconeogenesis compared to lower eukaryotes., Author summary Polycystic kidney diseases (PKD) are incurable inherited chronic disorders marked by fluid-filled cysts that frequently cause renal failure. A glycolytic metabolism reminiscent of cancerous cells accelerates cystic growth, but the mechanism underlying such metabolic re-wiring is poorly understood. PKD-like cystic kidneys also develop in mice that lack the RNA-binding protein Bicaudal-C (Bicc1), and mutations in a single copy of human BICC1 associate with renal cystic dysplasia. Here, we report that Bicc1 regulates renal gluconeogenesis. A screen for interacting factors revealed that Bicc1 binds the C-Terminal to Lis-Homology domain (CTLH) complex, which in lower eukaryotes mediates degradation of gluconeogenic enzymes. By contrast, Bicc1 and the mammalian CTLH complex regulated each other, and Bicc1 stimulated the accumulation of the rate-limiting gluconeogenic enzyme even in cells depleted of CTLH subunits. Our finding that Bicc1 is required for normoglycemia implies that renal gluconeogenesis may be important to inhibit cyst formation.

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.

Head-to-tail polymers of sterile alpha motifs (SAM) can scaffold large macromolecular complexes. Several SAM-domain proteins that bind each other are mutated in patients with cystic kidneys or laterality defects, including the Ankyrin (ANK) and SAM domain-containing proteins ANKS6 and ANKS3, and the RNA-binding protein Bicc1. To address how their interactions are regulated, we first determined a high-resolution crystal structure of a Bicc1-SAM polymer, revealing a canonical SAM polymer with a high degree of flexibility in the subunit interface orientations. We further mapped interactions between full-length and distinct domains of Bicc1, ANKS3, and ANKS6. Neither ANKS3 nor ANKS6 alone formed macroscopic homopolymers invivo. However, ANKS3 recruited ANKS6 to Bicc1, and the three proteins together cooperatively generated giant macromolecular complexes. Thus, the giant assemblies are shaped by SAM domains, their flanking sequences, and SAM-independent protein-protein and protein-mRNA interactions.

Summary Furin trafficking, and that of related proprotein convertases (PCs), may regulate which substrates are accessible for endoproteolysis, but tools to directly test this hypothesis have been lacking. Here, we develop targeted biosensors that indicate Furin activity in endosomes is 10-fold less inhibited by decanoyl-RVKR-chloromethylketone and enriched >3-fold in endosomes compared to the trans -Golgi network (TGN). Endogenous PC7, which resists this inhibitor, was active in distinct vesicles. Only overexpressed PC7 activity reached the cell surface, endosomes, and the TGN. A PLC motif in the cytosolic tail of PC7 was dispensable for endosomal activity, but it was specifically required for TGN recycling and to rescue proActivin-A cleavage in Furin-depleted B16F1 melanoma cells. In sharp contrast, PC7 complemented Furin in cleaving Notch1 independently of PLC-mediated TGN access. Our study provides a proof in principle that compartment-specific biosensors can be used to gain insight into the regulation of PC trafficking and to map the tropism of PC-specific inhibitors.

The secreted growth factor Activin-A of the TGFβ 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 II B 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 while 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 anti-tumor immunity. Paracrine Activin-A signaling emerges as a potential target for personalized therapies, both to reduce cachexia and to enhance the efficacy of immunotherapies.

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.

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.

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.

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.

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.

The CLIP biosensor reveals the spatiotemporal activity of the Nodal proprotein convertases Furin and Pace4 during embryonic development., 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.

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.

Nodal is a secreted protein of the transforming growth factor beta (TGFbeta) family that activates Smad2 and Smad3 transcription factors through complexes of type I and type II activin receptors and glycosyl-phosphatidylinositol-anchored coreceptors of the epidermal growth factor-like Cripto/FRL-1/Cryptic family. During early embryogenesis, it stimulates the proliferation of pluripotent progenitor cells and specifies, in a dosage-dependent manner, their subsequent allocation to distinct germ layers. Available evidence indicates that the signaling strength of Nodal is controlled at the level of endocytic uptake and turnover of activated receptor complexes in early endosomes, but insights into the underlying molecular mechanisms are still limited. In this review, I briefly survey literature on the trafficking of the related TGFbeta receptors, and I discuss recent findings indicating that endocytosis of Nodal is coupled to proteolytic processing of its precursor at the cell surface and that the maturation and internalization of Nodal need to be guided by Cripto to stabilize endosomal signaling platforms.

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 (Pcsk5Vcc/null) completely recapitulate the Pcsk5Vcc/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.

Endoderm differentiation and movements are of fundamental importance not only for subsequent morphogenesis of the digestive tract but also to enable normal patterning and differentiation of mesoderm- and ectoderm-derived organs. This review defines the tissues that have been called endoderm in different species, their cellular origin and their movements. We take a comparative approach to ask how signaling pathways leading to embryonic and extraembryonic endoderm differentiation have emerged in different organisms, how they became integrated and point to specific gaps in our knowledge that would be worth filling. Lastly, we address whether the gastrulation movements that lead to endoderm internalization are coupled with its differentiation.

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., Highlighted article: In the chick prechordal mesoderm, the Nodal precursor proNodal acts via a non-canonical route to inhibit BMP signalling and thus maintain Shh expression

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.

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.

International audience; 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.

In mammalian embryos, molecular cross-talk with extraembryonic tissues is essential to elaborate the primary body axes. Here, we review a series of reciprocal interactions that occur shortly after implantation in the uterus, and discuss how they are integrated in a complex signaling network to establish antero-posterior and dorso-ventral polarity. At the heart of this signaling network is the TGFbeta-related protein Nodal which acts on extraembryonic tissues to induce positive and negative feedback regulators at opposite poles of the egg cylinder. This likely results in an activity gradient which is instrumental to pattern the embryo proper.

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.

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.

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.

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.

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.

Cytotoxic T lymphocytes (CTL) kill target cells by perforin-mediated pore formation, induction of apoptosis by the Fas ligand, or both. It has been demonstrated that depolarized neurons can be induced to express major histocompatibility complex (MHC) class I antigens by interferon-gamma. Evidence for antigen-dependent CTL-mediated killing was obtained by transfecting neurons with MHC class I cDNA. The present study was designed to investigate the mechanisms of killing of cerebellar granule neurons depolarized by high K+ concentrations and thereby inducible for MHC class I antigen expression. We found that neurons express only low levels of Fas (APO-1/CD95) and are resistant to Fas ligand-mediated killing even when pretreated with cytokines. However, granules extracted from CTL as well as purified perforin induce almost complete lysis of neurons. These data suggest that CTL-mediated elimination of neurons involves the perforin, but not the Fas pathway of target cell killing.

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.

The present invention relates to puromycin-sensitive aminopeptidases, antibodies generated against said aminopeptidases, and to means and methods for the production thereof. The invention is also directed to nucleic acids coding for said aminopeptidases, and fragments thereof, to methods of obtaining such nucleic acid molecules or fragments of the invention, and to systems suitable for the expression of such nucleic acids. One particular aspect of this invention relates to deoxyribo- and ribo-oligonucleotides and derivatives thereof, as well as pharmaceutical preparations, therapies, diagnostics and commercial research reagents in relation to disease states which respond to modulation of the synthesis of the enzyme puromycin-sensitive aminopeptidase (PSA).

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;30:2423–2436

Cells of the macrophage lineage are ubiquitously distributed in the body, including the central nervous system. They represent an essential host defense system to protect from infections. However, recent evidence indicates that brain macrophages may also be responsible for tissue destruction, including loss of neurons and demyelination.

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.

The significance of the immune system in the recognition and rejection of tumor cells in general is highly debated. Nevertheless, glioblastoma may escape immune surveillance by producing transforming growth factor β2 (TGF-β2), which inhibits growth and function of the cellular elements of the immune system. Escape from TGF-β dependent immune suppression may be achieved by the loss of TGF-β receptors on lymphocytes or by the production of protease inhibitors; in glioblastoma cells the latter block the protease-dependent events of processing of latent TGF-β2 into the mature, bioactive form. In the absence of the production of bioactive TGF-β by glioblastoma cells, the immune amplification system of the central nervous system, which comprises astrocytes and microglia cells, is suggested to enable immune effector pathways to recognize and reject tumor cells. Tumor immune surveillance could even be facilitated by glioblastoma cells, which secrete cytokines including chemotactic factors and express major histocompatibility complex (MHC) class I and II antigens. This, however, would not only exclude secretion of TGF-β but would also require both the expression of tumor neoantigens and an absence of immune tolerance to these antigens. Both features have not yet been clarified.

For the past several years immunologists have been fascinated by a series of experiments showing that transforming growth factor beta (TGF beta) suppresses T- and B-lymphocyte growth as well as IgM and IgG production by B cells. Moreover, while exerting chemotactic activity on monocytes and inducing expression of interleukin-1 and interleukin-6 by these cells, TGF beta interferes with bacterially induced tumor necrosis factor alpha production, oxygen radical formation and the adhesiveness of granulocytes to endothelial cells. These mechanisms may provide the basis for the effect of TGF beta to prevent the microvascular changes associated with brain edema formation in bacterial meningitis. Given the potential of lymphocytes as well as macrophages to produce TGF beta 1, this cytokine may exert negative feedback signals on the immune response, provided the cytokine is processed from its latent form to the bioactive homodimer. Potent effects of TGF beta have been observed in experimental animals including the inhibition of the generation of virus-specific cytotoxic T cells and antiviral antibodies as well as the diminution of cellular infiltrates with decreased major histocompatibility complex class-II expression and CD8+ T cells in the tissue of virally infected animals. TGF beta may also be of importance in tumor immunology. By the production of bioactive TGF beta as detected in glioblastoma and acute T-cell leukemia, tumor cells may induce an immunodeficiency state and escape immune surveillance. In inflammation, monitoring of TGF beta in the tissue will bring light on the immune regulation in acute and chronic inflammatory diseases.

Because of the importance of neural recognition molecules expressed by glial cells to mediate interactions with neurons, growth factors and cytokines known to be functional during morphogenesis and in diseases of the nervous system were studied for their effects on recognition molecule expression by cultured immature and mature astrocytes from several brain regions. In cultures of immature astrocytes, transforming growth factors-beta 1 (TGF-beta 1) and -beta 2 (TGF-beta 2) and nerve growth factor (NGF) increased expression of the neural adhesion molecule L1, leading to a glia-mediated L1-specific increase in neurite outgrowth of dorsal root ganglion neurons on the astrocyte substrate. L1 expression induced by TGF-beta was inhibited by addition of antibodies to NGF, suggesting that TGF-beta influences L1 expression by modulating production of NGF by astrocytes. TGF-beta 1 and -beta 2 decreased expression of N-CAM by immature astrocytes. Since N-CAM expression was not affected by NGF and antibodies to NGF did not abolish the TGF-beta-induced decrease in N-CAM expression, NGF did not appear to be the mediator for regulating expression of N-CAM. Expression of the adhesion molecule on glia (AMOG) was not affected by any factor. NGF and TGF-beta 2 in latent form, but not TGF-beta 1 were found in the culture supernatants. Addition of interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), platelet-derived growth factor (PDGF), or basic fibroblast growth factor (bFGF) to the cultures did not change recognition molecule expression. REcognition molecule expression by mature astrocytes was not found to be modified by any of the factors tested. In view of the observation that levels of L1 and N-CAM expression correlated with the presence of TGF-beta 2 and NGF in the culture supernatants of immature astrocytes, an autocrine regulatory mechanism for recognition molecule expression by these cells is suggested to play a crucial role in regulation of neuron-glia interactions.

Summary: Phanerochaete chrysosporium produced several intracellular NADH:quinone oxidoreductases under agitated, nitrogen-limited cultivation conditions. One of the quinone reductases was purified and shown to have a molecular mass of 69 kDa by SDS-PAGE, while the molecular mass determined by gel filtration was 47 kDa. This reductase was separated by IEF into four protein bands, each with quinone reductase activity. The isoelectric points of the proteins were 5·7, 5·9, 6·0 and 6·3. The proteins reduced several quinones to the corresponding hydroquinones, but none of them was specific to any one of the quinones tested. Mycelial extracts of P. chrysosporium contained several more quinone reductases, with isoelectric points of 4·4, 4·7, 5·0, 5·3, 5·5 and 6·6. Quinone reductase activity could be induced by adding vanillic acid or 2-methoxy-1,4-benzoquinone to the growth medium in nitrogen-limited cultures and in carbon-limited cultures.

In vertebrates, specification of anteroposterior (A/P) and left–right (L/R) axes depends on TGFβ-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 betweennodal 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.