|
Fig. 2. Xctr1 knockdown inhibits differentiation and enhances morphogenesis. (A) Whole-mount immunohistochemistry of stage 32 embryos injected in the dorsal marginal zone at early cleavage stages with 250 ng of either Xctr1MO or Xctr1 5 base-pair mismatch (MM) morpholinos. Rescue experiments were performed by coexpression of a morpholino-insensitive Xctr1 construct (Xctr1*) in which the Xctr1MO-binding region was separated from the remainder of Xctr1 by 6 Myc epitope tags; somite formation was scored on a scale of 0 to 5 (0, complete, bilateral absence of 12/101 stain; 5, normal staining and somite morphology). Xctr1MO embryos were scored as 1.49 ± 0.3 (n = 41), and Xctr1MO + 500-pg Xctr1* embryos were scored as 2.01 ± 0.07 (n = 39). Xctr1* RNA (250 and 500 pg) were coinjected with Xctr1MO as indicated. (B) Effect of Xctr1 knockdown on FGF-mediated induction of Xbrachyury (Xbra) expression and its rescue by coexpression of 500 pg of Xctr1* RNA. Xctr1*, like Xctr1, induces expression of the neural marker NRP-1, albeit at higher doses than wild-type (data not shown). (C) Xctr1 knockdown inhibits Xbra expression in vivo. Whole-mount in situ hybridization analysis of gastrula-stage embryos using an antisense Xbra probe. Red-gal staining used as a lineage trace is demarcated with brackets; Xbra stain in Xctr1MO-injected embryos is indicated with arrows. (Upper) Marginal views. (Lower) Vegetal views. (D) Effects of Xctr1 knockdown on FGF- and activin-mediated morphogenesis of stage 20 animal caps. (E) Expression of dominant-negative Dishevelled (Xdd1) RNA inhibits Xctr1 knockdown-mediated elongation by FGF; 10 ng/ml FGF and 0.5 ng/ml (high) or 0.1 ng/ml (low) activin was added as indicated.
|
|
Fig. 3. Xctr1 regulates Ras-ERK signaling. (A) Coprecipitation of Xctr1, Laloo, and the docking protein, SNT-1/FRS2α. (B) Xctr1 knockdown inhibits Laloo-mediated SNT-1 phosphorylation (0.37 ± 0.11-fold reduction by Xctr1MO; n = 3). Signal represents the phosphorylation of up to six tyrosine residues on SNT-1 (10–12). (C) Xctr1 knockdown inhibits ERK phosphorylation (0.62 ± 0.27-fold reduction by MO; n = 4). Effects of injection of Xctr1MO and Xctr1MM on the FGF-mediated dual phosphorylation (dp) of the ERK MAP kinase in animal cap explants. Explants were cultured in the presence of FGF for 2 h before Western blot analysis; inhibition of ERK phosphorylation by Xctr1MO was not apparent for the first hour of FGF stimulation (data not shown). (D) Inhibition of activin-mediated dorsal mesoderm induction in animal cap explants by 1 ng of dominant inhibitory Ras (dnRas) RNA (1). (E) Rescue of Xctr1 morpholino-mediated dorsal mesoderm inhibition by coinjection of 40-pg constitutively active Ras (v-Ras) RNA (1). (F) Injection of 1 ng dnRas RNA does not enhance elongation of animal caps by FGF. Ras inhibition also inhibits elongation in FGF-treated caps from embryos injected with Xctr1MO (data not shown). (G) RT-PCR analysis of Xctr1 expression in early stage Xenopus embryos. (H) Whole-mount in situ hybridization analysis of Xctr1 expression. Stages (Upper) 4, 8 (animal views), 18 (dorsal view), and (Lower) 32 (lateral view) are shown. Pronephric tubules are indicated by arrowhead; 250-ng morpholinos were injected at early cleavage stages as listed, and 10 ng/ml FGF and 0.5 ng/ml activin were added to animal cap explants at stage 8 as listed.
|
|
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 β-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 μM 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.
|
|
Fig. 4. Ctr1 −/− ES cells exhibit prolonged pluripotency and defects in differentiation. (A) Morphology of wild-type, Ctr1 +/−, and Ctr1 −/− monolayers cultured for 4 days in the absence of LIF. Two lines each of Ctr1 +/− and Ctr1 −/− ES cells were assayed with similar results. One line derived from a wild-type blastocyst served as a control. (B) Flow-cytometric analysis of Flk-1 protein expression in wild-type, Ctr1 +/−, and Ctr1 −/− monolayers cultured for 4 days in the absence of LIF. Levels of c-Kit, a protein expressed in undifferentiated ES cells, hematopoietic stem cells, and germ cells, were not altered in Ctr1 +/− or Ctr1 −/− monolayers (41). Plots correspond to cells in A, as denoted by arrows. (C) RT-PCR analysis of wild-type (+/+), Ctr1 +/−, and Ctr1 −/− EB cultures. Differentiation is inhibited in the Ctr1 −/− EBs, whereas markers of pluripoptent ES and/or germ cell progenitors are enriched in these cultures. Morphology and expression of ES cell markers in Ctr1 +/− lines generally resembled that seen in wild-type lines. Expression of markers of differentiated fates was often slightly delayed and/or diminished relative to that seen in wild-type cells, suggesting a modest Ctr1 haploinsufficient phenotype (this figure and data not shown). In this regard, we note that Ctr1 +/− mice are viable and fertile (31, 32). β-actin was used as a loading control. (D) Proposed function of Ctr1 during early vertebrate development. (Upper) Model depicting a role for Xctr1 in the interpretation of FGF–receptor interaction as either a differentiation (Left) or a morphogenesis (Right) cue. (Lower) Comparison of Ctr1 function in amphibian embryonic and mammalian ES cell development. Red bars signify levels of Ctr1 activity.
|
|
Fig. 10. Xctr1 knockdown enhances mesodermal and neural morphogenesis. (A) Analysis of the timing of FGF addition on Xctr1 knockdown-mediated morphogenesis. Addition of FGF at midblastula stages (st 8.5), followed by removal (through washing) at midgastrula (st 10.5; Top) or late gastrula stages (st 12; Middle) is sufficient to induce elongation at late neurula stages (st 18); constant exposure of explants to FGF from midgastrula to late neurula stages, however, does not promote elongation (Bottom). (B) Xctr1MO inhibits markers of caudal neural fate; molecular markers are as described (1). To obtain neural tissue, animal caps were isolated from embryos injected with RNA encoding a truncated Bone Morphogenetic Protein 4 (BMP4) receptor (tBr), shown to generate anterior neural fate in ectodermal explants (2). FGF protein was added at stage 11, when explants are no longer competent to respond to mesoderm-inducing cues: FGF posteriorized tBr-induced neural tissue, inducing markers of the midbrain-hindbrain boundary (en-2), hindbrain (krox20), and spinal cord (Xcad3 and hoxB9), and decreased expression of the forebrain marker otx2. Coinjection of Xctr1MO inhibited expression of both general and caudal neural markers. (C) Xctr1MO enhances elongation of posterior neural tissue. (D) Xctr1 knockdown inhibits neuralization. Induction of the neural-specific markers NCAM, NRP-1, and Sox2 by tBr is inhibited by coinjection of Xctr1MO. Animal caps were isolated from uninjected embryos or from embryos injected with 2 ng tBr RNA and 250 ng of either Xctr1MO or Xctr1MM morpholinos at early cleavage stages as listed; 10 ng/ml FGF was added as indicated.
|
|
Fig. 9. Effects of Xctr1 knockdown in late neurula stage animal cap explants. RT-PCR analysis of stage 20 animal cap explants dissected at stage 8.5 and cultured in the presence or absence of added growth factors. (A) Effects of Xctr1 knockdown on FGF-mediated mesoderm induction at late neurula stages. (B) Effects of Xctr1 knockdown on activin-mediated induction at late neurula stages, and their rescue by coexpression of 500 pg Xctr1* RNA. Xctr1*, like Xctr1, induces expression of the neural marker NRP-1, albeit at higher doses than wild type (data not shown). (C) Xctr1 knockdown does not induce mesodermal or neural marker genes in animal cap explants at late neurula stages; Xctr1 knockdown also fails to induce neural crest markers in early neurula stage explants (data not shown). Animal caps were isolated from uninjected embryos, or from embryos injected with 250 ng of either Xctr1MO or Xctr1MM morpholinos at early cleavage stages, and cultured with 10 ng/ml FGF or 0.5 ng/ml activin as listed.
|
|
Fig. 8. Inhibition of neural and mesodermal development in Xctr1-depleted embryos. Whole-mount immunohistochemistry of embryos injected with Xctr1MM or Xctr1MO morpholinos, and stained with the somite-specific antibody 12/101 (brown) and the neural-specific antibody Xen-1 (blue); vibratome sections are shown at right. 12/101-positive tissue in Xctr1MO-injected morpholinos, when present, did not coalesce into the chevron pattern characteristic of somites in stage 33 Xenopus embryos. Neural tissue was disorganized and significantly diminished in Xctr1-depleted embryos; this effect may be secondary to the inhibition of dorsal mesoderm formation, and/or to defects in morphogenesis. 250 ng morpholino oligos were injected into the marginal zone of early cleavage stage embryos, as listed.
|
|
Fig. 7. Effects of control and Xctr1 morpholinos on the in vitro translation of Xctr1 and Xctr1* (Myc-Xctr1) RNA. Xctr1MO inhibits Xctr1 translation but not the translation of Xctr1* in a rabbit reticulocyte system. Xctr1 translation in vitro is not affected by the presence of either an unrelated control morpholino (CMO), or an Xctr1MO variant with five base-pair mismatches (Xctr1MM; data not shown).
|
|
Fig. 6. Xctr1 neuralizes ectoderm without inducing mesoderm. Injection of Xctr1 RNA induces expression of the neural-specific markers NRP-1 and Sox2 (but not NCAM), but does not induce the expression of the somite marker muscle actin, the notochord marker collagen type II at early tailbud stages (stage 22; Right), or the panmesodermal marker Xbrachyury at gastrula stages (stage 10.5; Left); 250 pg or 1 ng Xctr1 RNA was injected as indicated.
|
|
Fig. 5. Xctr1 associates with the Src kinase Laloo. (A) Alignment of vertebrate ctr1 genes. Putative transmembrane domains (TMD) 1-3 are boxed. (B) Identification of the Laloo-interacting domain of Xctr1 by coprecipitation of Xctr1 deletion constructs. Numbers denote amino acid residues of Xctr1. Immunoprecipitation of Flag-tagged Laloo was followed by Western blot analysis of Myc-tagged Xctr1 constructs. 1 ng each of RNA encoding Flag-tagged Laloo and Myc-tagged Xctr1 deletion constructs was injected, as listed, at early cleavage stages. (C) Detection of Laloo by Western blot analysis of coexpressed, immunoprecipitated, full-length Xctr1; 1 ng each of RNA encoding Myc-tagged Laloo and V5-tagged Xctr1 was injected, as listed, at early cleavage stages. (D) Schematic of the Xctr1 protein. The intracellular loop domain of Xctr1 (red) is sufficient for interaction with Laloo. Schematic after (22).
|
