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Fig. 1. The protein binding domain in human and frog Six1 are identical. A comparison of the amino acid sequence of the N-terminal Six Domain (SD, turquoise bar) and homeodomain (HD, black bar) of human and Xenopus Six1 compared to fly SO. Sequences in human versus Xenopus are identical. Sequences that are identical in all three animals are blocked in red, whereas those that differ in fly are blocked in white.
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Fig. 2. Six1 binds Pa2G4. (A) HEK 293T cells were transfected with six1-Flag and pa2g4-HA plasmids. Pa2G4-HA was detected by Western blot (WB) when Six1-Flag was immunoprecipitated with an anti-Flag antibody (IP). (B) HEK 293T cells were transfected with equimolar amounts of six1-Flag and eya1-myc plasmids. In addition they were transfected with varying relative amounts of pa2g4-HA plasmids: equal (1.0), half (0.5), quarter (0.25), eighth (0.125) or none (0). Eya1-Myc was detected by Western blot (WB) when Six1-Flag was immunoprecipitated (IP). Eya1 bound to Six1 when Pa2G4 levels were low (0.125 or 0.25 M ratios), but did not bind when Pa2G4 levels were increased (0.5 or 1.0 M ratios). Left-most lane shows no transfection.
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Fig. 3. Pa2G4 affects the transcriptional activity of two different reporters. Histograms representing the relative luciferase activity in cells transfected with the various constructs and luciferase reporters. The luciferase activity was normalized to Renilla expressed with a constitutive promoter. All values are represented as a ratio to the empty vector (CS2), error bars represent the standard deviation of 3 independent experiments performed on 3 different days. (A) In HEK 293T cells, Six1 and 2G4 significantly repress whereas Eya1 and Eya1+Six1 significantly activate expression of the ARE-luciferase reporter. Addition of Pa2G4 dampens this activation. (B) In HEK 293T cells, Six1 and Six1+Eya1 significantly activate and 2G4 significantly represses expression of the AP2-luciferase reporter. Addition of Pa2G4 dampens this activation. (C) In XTC cells, Pa2G4 significantly activates the ARE-luciferase reporter; AP2-luciferase expression also is enhanced but did not reach significance. *=p<0.05; **=p<0.01; ***=p<0.001.
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Fig. 4. Knock-down of endogenous Pa2G4 disrupts the formation of the neural border zone and its derivatives. (A) Xenopus embryos showing “typical” cleavage patterns at the 8-cell stage (8CS) and 16-cell stage (16CS). Diagram on right shows the 16CS fate map of which blastomeres are the major progenitors of the neural plate (NP), neural crest (NC), pre-placodal ectoderm (PPE) or epidermis (EPI) (based on Moody, 1987). Green asterisks indicate the sites of microinjection that target knock-down to the precursors of the neural crest and cranial placodes. (B)In situ hybridization assays of gene expression at neural plate or neural tube stages monitored after unilateral knock-down of endogenous Pa2G4. Right side of each image (except zic2-b) is the MO-injected side, and the left is the uninjected, control side that demonstrates the normal gene expression pattern. Genes expressed in the neural border zone are either undetected (arrow, msx1) or much smaller in size (bar, pax3). Genes expressed in the neural crest are either undetected (arrow, foxd3) or much more faintly stained (red arrow, sox9-a). Genes expressed in the PPE and placodes are greatly reduced in size/staining intensity (green arrows in sox9-a, sox9-b, six1, sox11, irx1-a, irx1-b). Concomitantly, the domain of neural plate/neural tube gene expression is expanded on the knock-down side of the embryo (red bars in sox11, irx1-a, irx1-b, sox2, zic2-a). Knock-down of endogenous Pa2G4 also results in ectopic expression of zic2 in the lateral epidermis, as well as maintained expression of foxd4l1 in the neural plate at stages long after it should be extinguished (blue arrows). (C) Knock-down of endogenous Pa2G4 disrupts gene expression in the otocyst and branchial arches. Otic genes are either reduced in intensity or undetected in the otocyst on the knock-down side (right image in each panel, red arrows). Left image in each panel shows gene expression on the control side of the same embryo. Green arrows denote reduction in gene expression in the neural crest-derived branchial arches. Two different embryos show that expression of otx2 in the ventral otocyst was either undetectable (right image) or reduced in size (black bar, middle image) in comparison to the control side (black bar, left image). (D) Measurement of the size of the otx2 expression domain shows that for those morphant embryos in which there was some otx2 expression, it is significantly smaller compared to the control side (* indicates p<0.001, paired t-test; bars indicate SEM). For all phenotypes in this figure, please see Table 1 for frequencies and the number of embryos analyzed.
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Fig. 5.
Loss and gain of Pa2G4 have different effects on the otocyst. (A) Two examples of pa2g4 morphants. In both, the dorsal-ventral diameter of the otocyst is smaller on the MO-injected side and gene expression levels are reduced. (B) In contrast, when Pa2G4 levels are increased by mRNA injection, the otocyst diameter is not significantly different from the control side of the same embryo, but gene expression levels also are reduced. (C) The dorsal-ventral diameters of pa2g4 morphants, measured in relative units by an eyepiece micrometer, were significantly smaller compared to the control side of the same embryo (n=13; p<0.00001, paired t-test), whereas they were not significantly different in embryos injected with pa2g4 mRNA (n=20; p>0.05).
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Pa2G4 gain-of-function enlarges the neural border zone and neural crest at the expense of placodes. (A) Three genes required for neural border zone formation are expanded when the level of Pa2G4 is elevated. Black bars indicate the width of the expression domain on the control side of an embryo, and red bars indicate the width in the same embryo on the side injected with 200 pg of pa2g4 mRNA, indicated by the pink lineage tracer. (B) Three genes required for neural crest formation are expanded (red bars for foxd3, sox9; red arrow for zic2) on the pa2g4 side of the embryos, compared to control sides (black) of the same embryo. For zic2, the neural plate also is expanded (red bar). For sox9, the otic placode domain (red arrow) is reduced. (C) Genes required for cranial placode formation are differentially affected by increasing pa2g4. For six1, a low dose (100 pg) broadens the PPE domain (red bar), whereas 200 pg reduces the intensity of expression (red arrow). The placode domains of sox11 and sox2 are enlarged at both doses (also see Table 2). The placode domain of irx1 is reduced at 100 pg, but either reduced (irx1-200-a) or expanded (irx1-200-b) with 200 pg. The neural plate expression of sox11 and irx1 are expanded (red bars) at either dose. The sox2 neural plate domain infrequently appeared broader, but quantitation verified that it is (see text). foxd4l1 expression is maintained in the neural plate long after it should normally be extinguished (red arrows). (D) Expression of pa2g4 in the precursor of the neural plate (see Fig. 4A) causes the ectopic expression of neural border (msx1) and neural crest (foxd3, zic2) genes in the neural plate midline (red arrows). (E) Co-expression of Six1 with Pa2G4 (left image) eliminates the ectopic neural plate expression of foxd3 (red arrow; cf. 5D) and ameliorates the significant expansion of foxd3 in the border zone (cf. 5B), suggesting that these phenotypes are due to interaction with another factor. Expression of Pa2G4 in the absence of Six1 (right image) preserves the ectopic neural plate (np) expression of foxd3 and broadly expands it past the border zone (bz) into the lateral epidermis (epi).
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Fig. 7.
Gain of Pa2G4 disrupts gene expression in the otocyst. Otic genes are either reduced in intensity or undetected in the otocyst on the injected side (right image, red arrows, pink lineage tracer). Left image shows gene expression in the otocyst (black arrows) on the control side of the same embryo. Green arrows denote reduction in gene expression in the neural crest-derived branchial arches. Note that the otocysts are present in all cases. For all phenotypes in this figure, please see Table 2 for frequencies and the number of embryos analyzed.
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Fig. 8.
Pa2G4 levels affect cell death and cell proliferation. (A) Knock-down of endogenous Pa2G4 by MO injection significantly reduces the number of apoptotic cells in the neural crest/PPE domain, as indicated by the TUNEL assay. Loss of Pa2G4 also significantly reduces the number of mitotic cells, as indicated by phosphorylated H3 (PH3) immunostaining. Embryos evaluated at stage 16. (B) Increasing levels of Pa2G4 by mRNA injection (100 pg, 200 pg, or 400 pg) has no significant effect on proliferation in the neural crest/PPE domain when embryos are evaluated at stage 18. Ctrl, control side; KD, knock-down side; inj, mRNA-injected side. *, p<0.05, paired t-test.
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Supplemental Figure 1: In Xenopus, there are two homeologues of pa2g4, one on the short chromosome (pa2g4S) and one on the long chromosome (pa2g4L). The nucleotide sequence surrounding the translational start site (ATG) of each is presented. Above these sequences (in blue) are the sequences of the two anti-sense Morpholino oligonucleotides (MO) that were used to knock-down endogenous Pa2G4 expression. MO#1 binds to both the short and long pa2g4 mRNAs within the open reading frame. MO#2 will bind to pa2g4S, but pa2g4L contains 5 mismatches (in red). The sequence of the rescue mRNA used to reverse the effects of MO knock-down is shown (pa2g4 rescue mRNA). This mRNA is insensitive to both MOs (mismatches in red) due to removal of the endogenous 5′UTR, insertion of the pCS2+ polylinker upstream of the ATG and insertion of an HA-tag downstream of the ATG that separates the MO binding from the open reading frame; MOs that bind more than 30 bases from the translational start codon do not block translation (www.gene-tools.com/choosing_the_optimal_target#TranslationalBlocking).
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Supplemental Figure 2: Validation of efficacy and specificity of pa2g4 MOs. (A) Expression of an mRNA that contains the native pa2g4S 5′ UTR and a 3′HA tag (5′UTR 2g4) expresses at high levels when injected into oocytes (duplicate lanes 2 and 3). Its expression is prevented by co-injection with MO#1+ MO#2 (duplicate lanes 4 and 5). uninj = uninjected oocyte lysate (lane 1). Numbers to the left indicate molecular weights. (B) Expression of an mRNA that is missing the native 5′UTR (rescue 2g4) expresses at high levels when injected into oocytes (duplicate lanes 1 and 2). Its expression is unaffected by co-injection with MO#1+ MO#2 (duplicate lanes 3 and 4). (C) Neural crest expression of foxd3 (blue stain on control [ctrl] side) is undetected on the MO-injected side (MOs) in a high percentage of embryos (n=66). (D) Neural crest expression of foxd3 (blue stain) is restored on the MO injected side by co-expression of an MO-insensitive mRNA (rescue) in a high percentage of embryos (n=24).
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