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Vertebrate Ctr1 Coordinates Morphogenesis and Progenitor Cell Fate and Regulates Embryonic Stem Cell Differentiation

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Vertebrate Ctr1 Coordinates Morphogenesis and Progenitor Cell Fate and Regulates Embryonic Stem Cell Differentiation Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation Tomomi Haremaki†, Stuart T. Fraser‡§, Yien-Ming Kuo¶, Margaret H. Baron‡§ʈ, and Daniel C. Weinstein†§†† Departments of †Pharmacology and Systems Therapeutics, ‡Medicine, and ʈMolecular, Cell, and Developmental Biology, Oncological Sciences, and Gene and Cell Medicine, and §The Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY 10029; and ¶Departments of Medicine and Pediatrics and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143 Edited by Igor B. Dawid, National Institutes of Health, Bethesda, MD, and approved June 7, 2007 (received for review February 14, 2007) Embryogenesis involves two distinct processes. On the one hand, cells pus copper transporter-1 (Xctr1) was identified as a factor that must specialize, acquiring fates appropriate to their positions (differ- physically associates with the tyrosine kinase Laloo, required for entiation); on the other hand, they must physically construct the FGF-mediated mesoderm induction [supporting information (SI) embryo through coordinated mechanical activity (morphogenesis). In Fig. 5] (9, 15). Microinjection of Xctr1 cRNA at early cleavage early vertebrate development, fibroblast growth factor (FGF) regu- stages gave rise to embryos with shortened, kinked axes and lates multiple embryonic events, including germ layer differentiation reduced or absent anterior structures, including eyes and cement and morphogenesis; the cellular components that direct FGF signaling glands (93%; n ϭ 46) (Fig. 1A). Xctr1-injected embryos underwent to evoke these different responses remain largely unknown. We at least some mesodermal differentiation, as demonstrated by show here that the copper transporter 1 (Ctr1) protein is a critical staining with an antibody (12/101) against a somite-specific epitope router of FGF signals during early embryogenesis. Ctr1 both promotes n ϭ A Left the differentiation and inhibits the morphogenesis of mesoderm and (96%; 46) (16) (Fig. 1 ). To better define the effects of neurectoderm in embryos of the frog Xenopus laevis, thereby coor- Xctr1 misexpression, ectodermal (animal cap) explant assays were dinating normal development. Signal sorting by Ctr1 involves the performed. Untreated animal caps normally ‘‘round up’’ and activation of the Ras–MAP kinase cascade and appears to be inde- develop as epidermis (Fig. 1B Upper Left); animal caps injected with pendent of its role in copper transport. Mouse embryonic stem (ES) Xctr1 RNA appeared indistinguishable from controls (data not cells deficient for Ctr1 (Ctr1؊/؊) retain characteristics of pluripotency shown). As expected, animal caps treated with high doses of the under conditions that favor differentiation in wild-type ES cells, TGF␤ ligand, activin, formed dorsal mesoderm (notochord and indicating a conserved role for Ctr1 during amphibian and mamma- somites) and elongated in a process known as convergent extension lian cell fate determination. Our studies support a model in which (17, 18); injection of a control RNA (GFP) had no effect on vertebrate Ctr1 functions as a key regulator of the differentiation elongation or gene expression (Fig. 1B Upper Center,Fig.1C, and capacity of both stem and progenitor cell populations. data not shown). Injection of Xctr1 RNA, however, markedly inhibited activin-induced elongation (Fig. 1B Lower Left). This Xenopus ͉ FGF ͉ pluripotent ͉ ERK ͉ Laloo effect was not secondary to a block of dorsal mesodermal differ- entiation because expression of the notochord marker collagen type recurrent theme in animal development is the prominent role II (19) was not affected in these explants; activin-mediated induc- Aheld by a small number of signaling molecules in the initiation tion of the somite marker muscle actin (20) was modestly reduced of events with a wide variety of biological outcomes. A dramatic or unaffected by Xctr1 misexpression (Fig. 1C, lanes 2 and 4, and example of this phenomenon occurs during early vertebrate em- data not shown). Xctr1 misexpression in animal caps cultured in bryogenesis, when fibroblast growth factor (FGF) mediates multi- saline induced the expression of the neural-specific marker NRP-1 ple developmental processes. During mesoderm induction, FGF (21) (Fig. 1D). This expression appears to reflect primary, rather triggers activation of a Ras–MAPK cascade (1–12). In contrast, than secondary, neural induction, in that neural induction by Xctr1, subsequent morphogenesis of the dorsal mesoderm is regulated by assayed at several time points, was not accompanied by mesoderm a noncanonical FGF pathway that is independent of Ras–MAPK 2ϩ induction (SI Fig. 6). Thus, Xctr1 misexpression neuralizes ecto- signaling and may involve stimulation of Ca release and/or dermal explants and disrupts gastrulation movements without activation of phospholipase C␥ (PLC␥) (13, 14). FGF, then, regu- inhibiting mesodermal differentiation. lates cell determination and morphogenesis via divergent signaling Ctr1, shown to mediate the uptake of cisplatin and other platinum cascades. BIOLOGY chemotherapy agents, encodes a putative three-pass transmem- The mechanisms underlying the distinct responses to FGF during DEVELOPMENTAL embryogenesis remain largely mysterious. FGF-mediated activa- brane protein thought to function in vivo as a high-affinity homotri- tion of the Ras–MAPK cascade during mesoderm development in meric transporter for reduced, monovalent copper in an energy- the frog Xenopus laevis is triggered by the coordinate phosphory- independent process (22–24). To determine the role of copper lation of the FRS2␣/SNT-1 docking protein by the FGF receptor transport in the regulation of cell movement, Xctr1 mutants were and by the Src family of nonreceptor tyrosine kinases (10–12). We generated based on mouse Ctr1 mutants shown to be defective in have identified the high-affinity copper transporter 1 (Ctr1) protein as a factor that physically interacts with the Src-related kinase, Laloo. Analysis of Ctr1 in frog embryos and in mouse embryonic Author contributions: T.H., S.T.F., M.H.B., and D.C.W. designed research; T.H., S.T.F., and stem (ES) cells demonstrates that Ctr1 is a critical regulator of tissue D.C.W. performed research; T.H. and Y.-M.K. contributed new reagents/analytic tools; T.H., S.T.F., M.H.B., and D.C.W. analyzed data; and D.C.W. wrote the paper. morphogenesis and of both stem and progenitor cell fate determi- nation. Our studies suggest that Ctr1 is required for the proper The authors declare no conflict of interest. interpretation and coordination of differentiation and morphoge- This article is a PNAS Direct Submission. netic cues during early vertebrate development. Abbreviations: EB, embryoid body; LIF, leukemia inhibitory factor. ††To whom correspondence should be addressed. E-mail: [email protected]. Results and Discussion This article contains supporting information online at www.pnas.org/cgi/content/full/ Protein interaction screens were performed to isolate novel com- 0701413104/DC1. ponents involved in interpreting FGF-stimulated functions. Xeno- © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701413104 PNAS ͉ July 17, 2007 ͉ vol. 104 ͉ no. 29 ͉ 12029–12034 Downloaded by guest on September 30, 2021 Fig. 1. Xctr1 misexpression inhibits morphogenesis in explants and embryos. (A) Whole-mount immunohistochemistry of stage 32 Xenopus embryos injected in the dorsal marginal zone at early cleavage stages with 1 ng of either Xctr1 or ␤-gal RNA. The 12/101 antibody is directed against a somite-specific epitope at this stage. (B) Effects of injection of RNA from wild-type and mutant Xctr1 on activin-mediated elongation of stage 20 animal caps; 1 ng of GFP RNA-injected animal caps were used as an injection control. Animal caps were explanted at stage 8; immediately after dissection, activin was added at a concentration of 0.5 ng/ml, and CuSO4 was added to achieve a concentration of 10 ␮M as indicated. (C–E) RT-PCR analysis of the effects of copper, wild-type, and mutant Xctr1 constructs on dorsal mesoderm induction by activin (stage 20) (C), neural induction (stage 20) (D), and metallothionein expression (stage 11) (E). (F) Xctr1 synergizes with FGF, but not activin, to induce Xbra expression (stage 13). (G) Copper-binding Xctr1 mutants, but not copper, synergize with FGF to induce Xbra expression (stage 13). Explants were treated with 0.5 ng/ml (C) or 0.1 ng/ml (F) activin protein or 10 ng/ml FGF protein (F and G) as listed; 1 ng of wild-type or mutant Xctr1 RNA was injected at early cleavage stages as listed. copper transport (25). The corresponding cRNAs (M2, M149) were zone injection of Xctr1MO resulted in a phenotype similar to that effective in blocking elongation of activin-treated animal caps at a seen in Xctr1 gain-of-function studies (compare Figs. 1A Left and range of concentrations similar to that of wild-type Xctr1 (Fig. 1B 2A Left). Few, if any, defects were seen after injection of a control Lower). Moreover, like wild-type Xctr1, expression of the mutant morpholino, Xctr1 mismatch (Xctr1MM), that differs from Xctr1 constructs induced NRP-1 expression and had little or no effect on by five base-pair substitutions (Fig. 2A). Strikingly, more than half the induction of dorsal mesoderm by activin (Fig. 1 C and D, of the Xctr1MO-injected embryos did not form somites, as dem- compare lanes 4, 5, and 6). Consistent with the observation
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