Identification of the WNT1 residues required for palmitoylation by Porcupine.

The post-translational palmitoylation of WNT morphogens is critical for proper signaling during embryogenesis and adult homeostasis. The addition of palmitoyl groups to WNT proteins is catalyzed by Porcupine (PORCN). However, the Wnt amino acid residues required for recognition and palmitoylation by PORCN have not been fully characterized. We show that WNT1 residues 214-234 are sufficient for PORCN-dependent palmitoylation of Ser224. Substitution of Ser224 with Thr, but not Cys, is tolerated in palmitoylation and biological assays. Our data highlight the importance of palmitoylation for WNT1 activity and establish PORCN as an O-acyl transferase for WNT1.

Bioaccumulation of benzo(a)pyrene from sediment by fathead minnows: effects of organic content, resuspension and metabolism.

The accumulation of 14C-benzo(a)pyrene (BaP) sorbed to sediment was examined in fathead minnows (Pimephales promelas) to compare uptake from sediment with a high organic carbon (OC) content (7.7%), to that with a low OC content (2%). Ingestion of sediments was quantified by co-labeling the sediment with 141Cerium, which was not assimilated by the fish. Results of this study indicated that (1) significantly greater quantities of BaP were dissolved in water over low-OC sediment, compared to water over high-OC sediment, (2) fish disturbed the sediment and increased the concentration of BaP in centrifuged (particle-free) water, (3) fish ingested significantly more of the low-OC sediment than high-OC sediment, perhaps in response to the lower food quality of the low-OC sediment, and (4) uptake of BaP from sediment ingestion contributed <3% of the total flux of BaP into the fish. Primarily as a result of the greater concentration of BaP in water, fish from the low-OC exposures had significantly higher rates of BaP accumulation. However, after 48 h the body burdens in these fish declined by 50%, likely due to the induction of MFO enzymes in response to accumulation of BaP. A smaller effect was apparent in the fish from the high-OC exposures, consistent with the lower dose of BaP they experienced. These results illustrate the complex, and sometime counterintuitive, interactions that affect the uptake and bioaccumulation of sediment-associated contaminants.

Comparative analysis of the expression patterns of Wnts and Frizzleds during early myogenesis in chick embryos.

During embryogenesis, the transduction of Wnt signals through Frizzled receptors is thought to play integral roles in myogenesis and somite patterning. However, little is known about which Wnt-Frizzled interactions are required for skeletal myogenesis. Thus, we sought to determine which Wnts and Frizzled exhibit expression patterns that are spatiotemporally consistent with the expression of two myogenic determination factors: Myf-5 and MyoD. To accomplish this, we first isolated partial cDNAs for six chick Frizzled orthologues and then compared the expression patterns of chick Frizzleds and Wnts to myogenic and somite patterning factors, such as Myf-5, MyoD, Sonic Hedgehog (Shh), Pax-1 and Pax-3 in Hamburger and Hamilton stage 10 chick. We used these data to generate a schematic composite of expression patterns at the level of the segmental plate and developing somites (stage V) that shows multiple Frizzled and Wnt transcripts expressed in tissues that are overlapping and adjacent to Myf-5 and MyoD expressing tissues.

Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo.

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.

Sfrp-1 and sfrp-2 are expressed in overlapping and distinct domains during chick development.

Secreted frizzled related proteins (Sfrps) are thought to bind and regulate Wnt activity through a cysteine rich domain that is highly similar to that of Frizzled receptors. To investigate possible roles for Sfrps in chick development, we have isolated partial cDNAs encoding Sfrp-1 and Sfrp-2 and have thoroughly characterized the expression patterns of both genes. Both sfrp-1 and sfrp-2 are expressed at all stages of development analyzed, ranging from Hamburger and Hamilton stage 4 through stage 32. Expression of both sfrp-1 and sfrp-2 is observed in mesodermal and ectodermal derivatives, while sfrp-1 is also found in endodermal lineages.

The dynamic expression pattern of frzb-1 suggests multiple roles in chick development.

The Wnt family of secreted proteins has been shown to have multiple roles in embryonic development. Wnt signals are thought to be propagated by binding to the cysteine-rich extracellular domain (CRD) of Frizzled, a seven-transmembrane-domain cell surface receptor. Secreted Frizzled-related proteins (generally denoted Frzb or Sfrp) possess a domain with a high degree of sequence identity and structural similarity with the CRD of Frizzled. Current data indicate that the cysteine-rich domain of secreted Frzb proteins can bind Wnt proteins, suggesting the possibility that Frzbs compete with membrane-bound Frizzled for Wnt binding and consequently act as competitive inhibitors of Wnt signaling. In order to gain a better understanding of the potential roles of Frzb-1 in chick development, we utilized the polymerase chain reaction to isolate a partial cDNA of the chick orthologue of frzb-1, cfrzb-1, and compared its expression pattern to that of Wnt-1, Wnt-3a, Wnt-5a, Wnt-7a, and Wnt-8c. Whole-mount in situ hybridizations have revealed three major phases of expression for cfrzb-1 in the developing chick. The earliest expression of cfrzb-1 is in cells fated to become neural ectoderm in streak-stage embryos. Expression of cfrzb-1 in the neural ectoderm continues up through stage 8. After stage 8, cfrzb-1 expression is gradually attenuated in the closing neural tube of the trunk and is concomitantly up-regulated in neural crest cells. Finally, cfrzb-1 appears in the condensing mesenchyme of the bones in both the limb and the trunk in stage 25+ embryos. Comparative analysis of the cfrzb-1 and the Wnt gene expression patterns suggests possible interactions between cFrzb-1 and all of the Wnt family members examined.

Cysteine-rich FGF receptor regulates intracellular FGF-1 and FGF-2 levels.

The cysteine-rich FGF receptor (CFR) is a 150-kD membrane-associated glycoprotein that specifically binds FGFs. CFR protein is not detectable at the cell surface and immunocytochemistry with anti-CFR antibodies demonstrates that CFR is concentrated in the Golgi apparatus. These data suggest CFR does not function as a plasma membrane FGF receptor. CFR expressed in chinese hamster ovary cells reduces the intracellular accumulation of exogenously applied FGF-1 and FGF-2. A mutant CFR lacking the juxtamembrane, transmembrane and intracellular domains is unable to alter intracellular FGF levels. Mutant CFR is detected throughout the cell, indicating that the domains absent in mutant CFR are required for appropriate subcellular localization and the regulation of intracellular FGF levels. Although the activation of plasma membrane receptors is necessary for cellular responses to FGFs, a requirement for intracellular FGF has also been proposed. The subcellular localization of CFR and its ability to regulate the levels of intracellular FGFs suggests that CFR may be involved in intracellular FGF trafficking and the regulation of cellular responses to FGFs.

Identification and characterization of a fibroblast growth factor (FGF) binding domain in the cysteine-rich FGF receptor.

Three distinct transmembrane glycoproteins bind fibroblast growth factor (FGF) family members. These include heparan sulfate proteoglycans, the tyrosine kinase-containing FGF receptors (FGFRs), and a cysteine-rich FGF receptor (CFR). The four FGFRs are thought to mediate FGF-signaling events but require the participation of the heparan sulfate proteoglycans to bind FGFs and transduce intracellular signals. However, a number of groups have proposed that FGF action requires events independent of FGFR activation. CFR, a high affinity FGF-binding protein, was first isolated from chicken embryos. To better understand the interactions between CFR and FGFs, we have constructed a series of CFR deletion mutants and CFR fragments. Analysis of these has identified a approximately 200-amino acid domain that constitutes a CFR FGF binding site. A CFR fragment of 450 residues, CFR290-740, binds FGF-2 with an affinity indistinguishable from the full-length molecule, whereas smaller fragments display greatly reduced FGF binding. Although CFR binds heparin with high affinity, an analysis of the heparin-CFR interaction failed to identify a linear sequence containing a heparin binding site. Two types of FGF binding sites were identified: an ionic strength and heparin-independent site that represents FGF binding to CFR290-740 and an additional FGF binding site that is heparan sulfate-dependent and sensitive to high ionic strength. This latter site is likely to bind FGF indirectly via heparan sulfate binding to CFR. FGF-2 peptides that encompass a sequence implicated in FGF-2 binding to FGFRs also block FGF-2 binding to CFR. Our data suggest that binding of FGFs to CFR and FGFRs is mutually exclusive, since the CFR FGF binding site does not require heparan sulfate, and similar regions on FGF-2 interact with both FGFRs and CFR.

Biochemical analysis of murine Wnt proteins reveals both shared and distinct properties.

The murine Wnt family of proteins consists of at least 12 members that possess significant amino acid homology. Current evidence suggests that these proteins are secreted cell-signaling molecules which are likely to have multiple roles during both embryonic development and oncogenesis. Although the biochemical properties of Wnt-1 have been thoroughly examined, less is known about the characteristics of other Wnt family members. We have compared the properties of six murine Wnt proteins (Wnt-1, Wnt-3a, Wnt-5a, Wnt-5b, Wnt-6, and Wnt-7b) transiently expressed in COS cells. All members enter the endoplasmic reticulum (ER) and are glycosylated. However, all six Wnt proteins are primarily retained in the ER in association with BiP, a resident ER protein that binds to improperly folded proteins and prevents their secretion and/or promotes proper folding. Although all Wnt family members examined are similarly processed, one notable difference was identified. Whereas addition of suramin to COS cell cultures significantly increases the levels of all six Wnts in the medium, the addition of heparin only influences the levels of Wnt-1, Wnt-6, and Wnt-7b.

Identification of a cysteine-rich receptor for fibroblast growth factors.

The fibroblast growth factor (FGF) family consists of seven members whose activities are thought to be mediated by multiple receptors. Here we describe the cDNA cloning, expression, and characterization of a cysteine-rich FGF receptor (CFR) that is distinct from previously identified FGF receptors. The deduced amino acid sequence for CFR suggests that it is an integral membrane protein containing a large extracellular domain comprising 16 cysteine-rich repeated units and an intracellular domain of 13 amino acids. No reported sequences exhibit significant homologies to either the repeated extracellular motif or to the entire CFR amino acid sequence. Several CFR transcripts are present in embryonic chick tissue, suggesting that CFR undergoes alternate mRNA splicing or that related genes are present. Chinese hamster ovary cells transfected with the CFR cDNA express a 150-kDa polypeptide that binds FGF-1, FGF-2, and FGF-4 but does not bind several non-FGF family members. The high degree of evolutionary conservation among vertebrate CFRs and its ability to bind three different FGFs with high affinity suggest that this unique receptor plays an important role in FGF biology.

Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick.

A receptor for acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) was isolated from 7-day embryonic chick. Chromatography of solubilized membrane proteins on wheat germ agglutininagarose and aFGF-Sepharose yielded three major polypeptides migrating at 150, 70, and 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides were eluted from aFGF-Sepharose with either 1.0 M NaCl or 100 micrograms/ml heparin, but were not retained on underivatized Sepharose. Cross-linking of 125I-aFGF or 125I-bFGF to either crude membrane preparations or to purified fractions yielded a 165-kDa complex, suggesting the existence of a 150-kDa FGF receptor after subtraction of approximately 15 kDa for 125I-FGF. Addition of excess aFGF or bFGF competed for binding of either 125I-aFGF or 125I-bFGF to FGF receptor preparations. Purified FGF receptor fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and incubated with 125I-aFGF or 125I-bFGF in order to identify FGF-binding polypeptides. Bound 125I-aFGF and 125I-bFGF were displaced by aFGF and bFGF, but not epidermal growth factor, consistent with the identification of the 150-kDa polypeptide as a receptor for acidic and basic FGF. Treatment of purified FGF receptor fractions with N-glycanase demonstrated that the 150-kDa polypeptide contained approximately 10 kDa of N-linked oligosaccharide. The apparent molecular mass of the 150-kDa polypeptide was unaffected by treatment with heparitinase, indicating that the 150-kDa polypeptide is not a heparan sulfate proteoglycan. Together, these data suggest that the 150-kDa polypeptide is a FGF receptor that may mediate the biological activities of aFGF and bFGF.