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Figure 1. PKC Regulates LR Development during Early Gastrulation early gastrula stage during which a cPKC is required for (A) Percentage of reversed heart looping in embryos injected with cell-permeable PKC inhibitors into the blastocoel of stage 10.5 embryos. Myristoylated peptides ( , , ) block translocation and function of conventional (cPKCs), novel (nPKCs), or atypical (aPKCs) PKCs, respectively. (B) Percentage of reversed heart looping in embryos injected with the myristoylated peptide that blocks cPKCs. The blastocoel was injected at several stages ranging from blastula to late gastrula. (C) Percentage of reversed heart looping in embryos injected with mRNA encoding dominant-negative (dn) PKC constructs for each of the cPKCs ( , , or ). n 100 for each injection. (D) Representative in situ hybridizations of Xnr1 in the lateral plate mesoderm illustrating the most prevalent expression classes: uninjected embryos display Xnr1 expression on the left, and myristoylated peptide injected embryos display bilateral Xnr1 expression. Panels are dorsal views with anterior to the left.
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Figure 2. PKC Asymmetrically Regulates LR Development and Is
Required in Right-Side Ectoderm
(A) Percentage of reversed heart looping in embryos injected with
mRNA encoding dnPKC . The schematic animal-pole view of a 32-
cell embryo illustrates the percentage of reversed heart looping
observed when the corresponding cell was injected. Red indicates cells.
high reversal rates, yellow is intermediate reversal rates, and gray
is background rates. Top is ventral. n 69 for each injection. (B)
Western blots of homogenates from stage 11 uninjected and mor-
pholino (mo)-treated embryos probed with antibodies to PKC and
B-catenin. (C) Percentage of reversed heart looping in embryos injected
with anti-PKC morpholinos. Schematic is the same as in
(A), except a 16-cell embryo is illustrated. n 60 for each injection.
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Figure 3. Endogenous Syndecan-2 Is Phosphorylated in the Right Ectoderm during Gastrulation (A) Antibody specificity. Dot blots of chemically phosphorylated syndepeptides
used in the generation of phosphospecific antibodies. Ab-breviations: R, random, nonspecific peptide; S2, unphosphorylated cGERKPS150S151AVY; P1, phosphorylated at S150; P2, phosphorylated at S151; PP, phosphorylated at S150 and S151. Antibody S2ALL recognizes
all forms of syndecan-2 peptide regardless of phosphorylation
state. Antibody S2P recognizes only the phosphorylated forms of
syndecan-2. (B) PKC inhibitor blocks syndecan-2 phosphorylation.
Embryos were injected with -myristoylated peptide (see Figure 1)
at stage 9.5, 10.5, or uninjected. At midgastrula (stage 11), extracts
were immunoprecipitated with antibody 6G12, deglycosylated, and
blotted with antibodies that recognize phosphorylated syndecan-2
(left). Homogenates from the same embryos were probed for
-catenin as an extract loading control (right). (C) Syndecan-2 is
phosphorylated on the right side. Embryos were dissected into left
and right halves at stage 11, immunoprecipitated with antibody
S2ALL, deglycosylated, and blotted with antibodies that recognize
all forms of syndecan-2 (left) or only phosphorylated syndecan-2
(right). In addition to showing the phosphorylated upper band (ar-
rowhead), the S2ALL Western blot serves as a loading control for
the immunoprecipitation. (D) Extracts fromembryo halves were pro-
cessed as in (C) but were immunoprecipitated with the syndecan-2- specific
antibody 6G12. Immunoprecipitates were probed with
phosphospecific syndecan-2 antibody (left). Homogenates from the same
embryo halves were probed for -catenin as a control (right).
(E) Stage 11 whole-mount immunocytochemistry, using antibodies
that recognize all (left) or phosphospecific (right) forms of
syndecan-2.Migrating leftmesoderm(L) is in contact with ectoderm
that expresses nonphosphorylated syndecan-2, and right mesoderm (R) is in contact with ectoderm expressing phosphorylated
syndecan-2. These illustrations are representative of over 100 embryos examined.
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Figure 4. Both Phosphorylated Syndecan-2 on the Right and Nonphosphorylated Syndecan-2 on the Left Are Required for Normal
LR Development
Percentage of reversed heart looping in embryos injected with a
phosphodeficient syndecan-2 (A) or a phosphomimetic syndecan-2
(B) is shown on the right side of each panel. The left side of each
panel is a diagramof the syndecan-2 constructs used in each experi
ment. The relative positions of the serine-to-alanine (A) or serine-to-
glutamine (B) mutations are shown on the cytoplasmic domain.
Wavy lines indicate the glycosaminoglycans attached to the
syndecan-2 extracellular domain. See Figure 2 for an explanation of the
embryo illustration. n > 55 for each cell.
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Figure 5. PKC Functions Upstream of Syndecan-2 in the Same LR Developmental Pathway (A) Inhibition of PKC synthesis blocks phosphorylation of synde-can-2. Western blot of untreated or PKC -morpholino injected stage 11 embryos probed with antibodies that recognize all forms of syn-decan-2 (left) or only phosphorylated (right) syndecan-2. Extracts were
processed as in Figure 3. Antibody S2ALL serves as a loading control. (B) Epistasis analysis of PKC and syndecan-2. Percentage of reversed heart looping in embryos injected on the right side with morpholinos to PKC (moPKC ), phosphomimetic syndecan-2 (S2-EE), or both the morpholino and the phosphomimetic syndecan-2. n 89 for each injection. (C) Percentage of reversed heart looping in embryos injected with dominant-negative PKC (dnPKC ), S2-EE, or both. n 31 for each injection. Expression of phosphomimetic syndecan-2 rescues LR development in embryos that are deficient for PKC .
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Figure 6. Syndecan-2 Is an Early Cell-Nonautonomous Transducer of LR Axis Patterning Information (A) Timeline of Xenopus LR development, illustrating when each molecule has been shown to function (H K ATPase, gap junc-tions, PKC , and syndecan-2) or appear (asymmetric phosphorylation states of syndecan-2, LR dynein (LRD), node monocilia, and asymmetric expression of nodal and lefty in lateral plate mesoderm).
See text for references. (B) A model for PKC -syndecan-2 function
in LR development. PKC is either activated exclusively on the right
or inhibited exclusively on the left, resulting in phosphorylation of
syndecan-2 only in the right ectoderm. The LR asymmetry is trans-
duced through syndecan-2, leading to asymmetric patterning of the
mesoderm where nodal, lefty, and pitx2 are eventually expressed
only in the left mesoderm.
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Xenopus PKCγ Cloning and Characterization
Based on human, bovine, mouse, and rat PKCγ, two independent rounds of Xenopus PKCγ cloning both produced an equal number of clones that encode for a full-length or a short Xenopus PKCγ. The short PKCγ appears to be a 5â² end splice variant of the full-length PKCγ, such that the short PKCγ has a different 5â²UTR and alternative translation start site. Three observations suggest that both splice variants of PKCγ are functional in LR development. First, the 5â² RACE was done using Invitrogenâs GeneRacer Kit in which an RNA oligo is ligated only to capped RNAs. Second, in order to observe a phenotype using PKCγ morpholinos, a morpholino against each translation start site had to be coinjected; either morpholino by itself had no effect (data not shown). Finally, the LR phenotype resulting from the morpholinos could be rescued by full-length mouse PKCγ (see text).Sequences for short (A) and full-length (B) 5â² ends of Xenopus PKCγ. Morpholinos used correspond to the underlined sequences. Alignment (C) showing conservation between both Xenopus PKCγ splice variants, mouse PKCγ, and Xenopus PKCα and PKCβ sequences. Only the 5â² region for all sequences is shown (corresponding to amino acids 1â88 of mouse PKCγ). Dark gray boxes show amino acid identity while light gray boxes represent similarity. Genbank accession numbers (pending).
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Syndecan-2 Antibody Specificity
As proteoglycans, syndecan preparations that have not been deglycosylated run as smears near the top of the gels. In order to deglycosylate, proteoglycans have traditionally been purified by affinity chromatography or immunoprecipitation and sugars removed by elimination with nitrous acid (as we have done in the main text) or digestion with enzymes like heparitinase. Although, as stated in the Experimental Procedures, no bands appear on the gel other than those we show, we wanted to demonstrate the syndecan-2 antibody specificity by straight Western blot. In order to do this, we homogenized whole embryos in PBS with protease and phosphatase inhibitors, added 20 mU heparitinase, and incubated the homogenate overnight at 37°C. Triton X-100 was subsequently added to a 1% final concentration, and the homogenate cleared and processed as detailed above. Western blots of hepartitinase digested (Hep) and undigested (Undig) homogenates blotted with αS2ALL (left panel) and αS2P (right panel) to demonstrate the antibody specificity.
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