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Display additional annotations [+]
| Gene |
Clone |
Species |
Stages |
Anatomy |
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ctnnb1
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laevis
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NF stage 39
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mesoderm
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endoderm
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stomach
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epithelium
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left
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[+]
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tuba4b
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laevis
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NF stage 39
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mesoderm
,
endoderm
,
stomach
,
epithelium
,
left
,
[+]
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Fig. 4. Pitx2c controls epithelial morphogenesis in left stomach wall. Frog embryos were injected with control
morpholino (control-MO; A,E,I,M,Q) or Pitx2c-MO (B,F,J,N,R) targeted to the left side of the stomach, or injected with Pitx2c- GR mRNA (C,G,K,O,S and D,H,L,P,T) targeted to the right side. (See Fig. S6A,B for morpholino validation.) In injected
embryos (A-D), the greater curvature of the stomach at stage 42 is indicated by an arrowhead (A,C); absence of curvature is
specified by an asterisk (B,D). Sections through stage 39 stomachs (E-T) were stained for β-catenin (βcat; red; E-L),
α-tubulin (αtub; green; M-P) or atypical PKC (aPKC; red; Q-T). GFP mRNA was coinjected as a lineage tracer to confirm proper targeting (green; E-H). MO depletion on the left (F) or ectopic activity of Pitx2 on the right (H) results in a loss of asymmetry
within the stomach compared with controls (E,G, respectively). In addition, compared with control-MO injected embryos, in which αtub and aPKC are concentrated at the apical surface of the left stomach wall (arrowheads in M,Q, respectively), MO
depletion of Pitx2c disrupts epithelial architecture (brackets in J,N,R). Meanwhile, dexamethasone induction of Pitx2c activity in the right wall polarizes stomach endoderm, as indicated by ectopic regions of polarized epithelial architecture (arrowheads in L,P,T), correlating with ectopic αtub (P) and aPKC (T), which are not observed in right wall of uninduced controls (K,O,S). Scale bars: 500 μm in A-D; 75 μm in E-H; 50 μm in I-T. L, left; R, right. |
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Figure S5. CRISPR-Cas9 mediated editing of pitx2c gene perturbs stomach asymmetry. Xenopus embryos
were injected with 2 ng synthetic Cas9 mRNA plus 300 pg control tyrosinase (tyr) gRNA (A) or pitx2c gRNA
(B), and allowed to develop until stage 42. The graph (C) indicates the percentage of embryos in which the
greater curvature of the stomach is normal (arrowhead in A) or reduced/absent (* in B) after injection with the
tyr gRNA or two different pitx2c gRNAs (#1 and #2). D) Genomic DNA from 10 embryos injected with each
gRNA was pooled and PCR amplified using exon 1-specific primers, and then tested for CRISPR/Cas9-induced
mutations by T7 endonuclease I assay. The asterisk (*) indicates the 500 bp amplicon not cut in un-injected or
tyr gRNA-injected control embryos. Arrowheads indicate bands resulting from mismatches (inferred mutations)
in amplicons cleaved by T7 endonuclease I. E) Sequencing of a subset of individual clones validates the
presence of deleterious mutations in pitx2c gRNA injected embryos. For pitx2c gRNA #1, 9/17 mutations were
likely nulls and 3/17 were predicted to result in compromised function; for pitx2c gRNA #2: 2/18 mutations
were likely nulls and 4/17 were likely to result in compromised function. F-N) Sections through stomachs of
Cas9 control and pitx2c gRNA (#1) injected embryos (Stage 39) were stained for integrin (green) and false
color-coded as in Fig. 1 to highlight the relevant tissue layers (RE, right endoderm; LE, left endoderm; RM,
right mesoderm; LM, left mesoderm). Controls show normal asymmetric expansion of the left stomach wall
(F), but this is eliminated in embryos injected with pitx2c gRNA (G-H). Normal asymmetries in the lengths of
the left and right stomach walls (I), and the widths of the endoderm (Endo) and mesoderm (Meso) layers (J-K),
are also significantly perturbed by CRISPR-Cas editing of pitx2c; * denotes p<0.01. Compared to controls (L),
left endoderm tissue architecture is severely disrupted, and apicobasal polarity is reduced (M), and/or
disorganized (N) in embryos with CRISPR-mediated mutations in pitx2c; arrowheads indicate the expression of
the apical marker aPKC (red). Scalebars= 100 M. |
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Figure S4. Markers of apicobasal polarity become left-right asymmetric in the developing stomach. The
intensity of several markers of apical polarity, including aPKC, Par3, E-cadherin (E-cad), alpha-tubulin (a-tub) and
gamma-tubulin (g-tub) were measured at the left and right surfaces of the frog stomach lumen at the indicated stages using image J (A). The level of apical enrichment is represented as a ratio of left and right intensities measured
in at least 3 sections of 3-5 different embryos. The left/right (L/R) ratios of all apical markers become
significantly different by stage 39, while the L/R ratios of Beta-catenin (B-cat) and integrin are not significantly >1.
B-J) High magnification images of sections through the Stage 39 stomach stained forB-catenin B-cat; red; BD),
alpha-tubulin (atub; green; E-G), or atypical PKC (aPKC; red; H-J). Compared to control embryos (B, E, H),
in which a-tub and aPKC are concentrated at the apical surface of the left stomach wall (arrowheads in E, H,
respectively), MO depletion of Pitx2c (C, F, I) disrupts epithelial architecture (arrows in I). Meanwhile,
dexamethasone-induction of Pitx2c activity (D, G, J) in the right wall ectopically polarizes the stomach
endoderm, as indicated by ectopic regions of polarized epithelial architecture, correlating with enriched tub
and ectopic aPKC at the apical surface (arrows in G, J). Scalebars = 25uM. Left (L), Right (R). |
