Yoder MD , Gumbiner BM .

AR050960-05 NIAMS NIH HHS , R01 GM52717 NIGMS NIH HHS , R01 GM052717-14S1 NIGMS NIH HHS , R01 GM052717-15 NIGMS NIH HHS , R01 GM052717-16 NIGMS NIH HHS , R01 GM052717 NIGMS NIH HHS , T32 AR050960 NIAMS NIH HHS

	Figure 1. A newly identified allele of AXPC is predominantly expressed during early development. A: Comparison of the sequence of the two alleles of AXPC surrounding the ATG start codon. MO-1 refers to a previously published construct (orange). MO-2 is a newly designed morpholino (green). Red box indicates the ATG start codon. *Differences between the sequences. B: Western blot detection of both exogenous and endogenous expression of AXPC in lysates from embryos collected at the indicated stage. Embryos over-expressing AXPC (lane: AXPC) were collected at stage 12.5. C: Western blot of endogenous AXPC in embryo lysates at stage 12.5 injected with the indicated amount of control morpholino or AXPC morpholino. For co-injections, morpholino-1 and morpholino-2 were mixed in equal parts to give the indicated final amount. In B and C, AXPC was detected by Western blotting with anti-AXPC antibody. Each lane represents the equivalent of 1 embryo.Download figure to PowerPoint
	Figure 2. Morpholino to the second AXPC allele causes developmental axial defects. A–D: Embryos were injected on one side of a 2-cell embryo with 10, 20, or 40 ng (shown) of morpholino along with a Dextran-Ruby tracer. Embryos were cultured and observed at stages 22 (C,D) and 30 (A,B). Fluorescent Dextran-Ruby (red) images were superimposed onto brightfield images in C and D. E: Embryos were scored at stage 30 as a strong (>45°), mid/weak (0°< x <45°), or no (0°) phenotype. Anterior is up. F,G: Whole mount immunofluorescence on embryos bisected sagitally at stage 30, stained with anti-AXPC (green) and anti-fibronectin (red). Asterisks denote side of morpholino injection. Confocal images obtained from ventral aspect at 600× magnification. Scale bar = 50 μm.Download figure to PowerPoint
	Figure 3. Loss of AXPC expression perturbs notochordal morphogenesis. A: Two-cell stage embryos were injected bilaterally in the dorsal marginal zone with 40 ng control morpholino (a), 20 or 40 ng AXPC morpholino (c,d) or 1 ng AXPC mRNA (b) and cultured until uninjected controls reached stage 12.5. Stage was determined by blastopore size and percentage was calculated by: no. in given stage/total no. embryos (A', n>50). B: Whole mount immunofluorescence of bisected embryos injected with either control morpholino (CtlMO, a–c) or AXPC morpholino-2 (AXPC MO2, d–i). Sections were stained to detect C-cadherin to outline cell borders (a,d,g) and fibronectin to delineate tissue boundaries (b,e,h). Dextran tracer can be observed as red in c, f, and i. Arrowhead in h marks a localized loss of FN staining. Confocal images acquired at 200× magnification. Scale bar = 150 μm.Download figure to PowerPoint
	Figure 4. AXPC does not mediate cell sorting. A–C: In cell dispersal assays, embryos were injected into one cell at 32-cell stage with nuclear-GFP alone (A), GFP+PAPC (B), or GFP+AXPC (C) and cultured to stage 9. Scale bar = 0.5 mm. D–F: Reaggregation assay performed using blastomeres isolated from excised animal caps expressing RFP, GFP, AXPC-mCherry, and PAPC+GFP. Cells were mixed as indicated and images were acquired once aggregates formed (5–6 hr). Scale bar = 1 mm. G,H: AXPC constructs have functional activity in perturbing notochordal morphogenesis. Sagittal cryosections of stage-12.5 embryos injected dorsally at 2-cell stage, with 1 ng RFP (I) or 1 ng AXPC (J). C-cadherin, fibronectin (FN), dextran-TRITC (red). Confocal images taken at 200× magnification. Scale bar = 150 μm.Download figure to PowerPoint
	Figure 5. AXPC is specifically required for specification of axial mesoderm but not general mesoderm induction. A: Embryos were injected bilaterally in the dorsal marginal zone at the 2-cell stage with 40 ng of either control (CtlMO) or AXPC morpholino (AXPCMO), then cultured until stage 12.5 and prepared for in situ hybridization with either chordin or MyoD. A': Embryos were scored by using the categories of “normal,” “reduced,” and “absent” to describe the staining intensity. Examples of normal and reduced are depicted (a,c or b,d, respectively) (n > 30). Anterior is up. B: Embryos were prepared as described above for the detection of chordin at stage 10.5. B': The categories “strong,” “mid,” and “weak” represent the degree of chordin staining used to score the control and AXPC MO–injected embryos. The bottom image is a higher magnification of the dorsal lip (boxed region) (n>30). C: RT-PCR analysis of animal caps from embryos injected with either control or AXPC MO and treated with or without 100 ng activin to induce mesodermal gene expression. Data were normalized to histone 4 (×H4). D: Mesoderm is not respecified to nueroectoderm or endoderm due to loss of AXPC. Embryos were injected as described in A and probed for Xbra, Sox17 (endoderm), or Sox2 (neuroectoderm). Asterisks denote the blastopore. Arrowhead indicates dorsal lip. Scale bar in A and B = 250 μm.Download figure to PowerPoint

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