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Figure 2. Sox4 or Sox11 loss of function results in an abnormal eye development.Effect of Sox4 (A) and Sox11 (B) depletion on eye development. The injection of Sox4 or Sox11 MO leads to smaller, deformed, and severely deformed eyes (white arrows) as illustrated in a dose dependent manner. A Control MO injection had no effect on eye development. Detailed views demonstrated defects in the formation of the RPE (red arrows). Vibratome sections showed that Control MO injection had no influence on the formation of the retinal layers and the RPE as well whereas depletion of Sox4 or Sox11 resulted in an abnormal RPE and retinal layering (red arrows). Quantitative representations are given. The eye phenotype of Sox4 or Sox11 down-regulation was significantly rescued by the co-injection of either SOX4 or sox11 RNA. GCL = ganglion cell layer, INL = inner nuclear cell layer, n = number of independent experiments, N = number of injected embryos analyzed, ONL = outer nuclear cell layer, RPE = retinal pigment epithelium. Error bars indicate standard error of the means (s.e.m.), * P≤0.05, *** P≤0.001.
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Figure 3. Sox4 depletion interferes with retinal lamination.Unilateral injection of 40 ng Sox4 or 40 ng Sox11 MO had no effect on the primary formation of most retinal cell types as shown by the expression of specific marker genes in mild eye phenotypes at stage 41. Only ganglion cells disappeared upon depletion of Sox4 or Sox11 in the severe eye phenotype. Many retinal cells were displaced (red arrowheads). Especially photoreceptor cells are displaced into inner layers of the retina (red arrowheads). In addition, the RPE is affected (black arrowheads). The uninjected (uninj.) sides revealed normal retinal lamination. For each marker gene, several embryos of different independent experiments were analyzed and showed a similar phenotype. Scale bar indicates 100 µm.
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Figure 4. Proneural genes are not affected upon loss of Sox4 or Sox11.At stage 32, expression of proneural genes was not changed after loss of Sox4 or Sox11. Scale bar indicates 100 µm.
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Figure 5. Lens development upon Sox4 or Sox11 depletion.Lens development was normal in Sox4 depleted embryos as shown by marker gene expression at stage 36. Loss of Sox11 resulted in a mild change of marker gene expression. n = number of independent experiments, N = number of injected embryos analyzed, n.s. = not significant, * P≤0.05,** P≤0.01.
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Figure 6. Eye-specific marker genes are unaffected upon loss of Sox4 or Sox11 function.A and B: Depletion of Sox4 function had no influence on marker gene expression at stage 13. Loss of Sox11 led to a slight decrease of pax6 expression. C and D: At stage 23, Sox4 depleted embryos revealed a reduced expression of rax, pax6, sox3 and en2. Emx1 and egr2 expression was not affected. In contrast, all marker genes were mildly affected upon Sox11 knock-down. B and D. Quantitative representations are given. n = number of independent experiments, N = number of injected embryos analyzed, n.s. = not significant, * P≤0.05,** P≤0.01.
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Figure 7. Loss of Sox4/11 function leads to cell apoptosis in the developing eye.A: TUNEL staining of Sox4 or Sox11 depleted embryos at stage 32. The areas where apoptotic cells have been counted are highlighted by a dotted circles and increased cell apoptosis was labeled by arrows. B: Quantitative representation of the TUNEL staining shown in A. n = number of independent experiments. **** P≤0.0001. C: Increased caspase 3/7 activity after loss of Sox4 or Sox11 at stage 32. Values represent relative light units (RLU) normalized to Control MO injected embryos. n = number of independent experiments. Error bars indicate standard error of the means (s.e.m.). D: Unilateral injection of 40 ng Sox4 or Sox11 MO led to increased caspases 3/7 activity while uninjected sides or Control MO injected embryos were not affected. Values represent relative light units (RLU) normalized to the uninjected side. n = number of independent experiments. Error bars indicate standard error of the means (s.e.m.). E: Increased cell apoptosis upon loss of Sox4 and Sox11 was rescued by tp53 inhibition. F: A quantitative representation of the results in E is given. n = number of independent experiments, N = number of injected embryos analyzed. G: Crosssections of Sox4 or Sox11-depleted embryos demonstrating mitotic cells (blue, pH3 staining). Black arrowheads point to the MO injected side. H: A quantitative representation of the data in F is given. Sox4 or Sox11 depletion had not significant (n.s.) effect on cell proliferation in the eye at stage 32. n = number of independent experiments. Error bars indicate standard error of the means (s.e.m.), n.s. not significant, * P≤0.05, **** P≤0.0001.
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Figure 1. Spatial expression of sox4 and sox11 during Xenopus laevis embryogenesis.
A–N sox4 expression, A’–N’ sox11 expression. The cement gland is depicted by a dotted circle. A,A’: At stage 1, sox4 and sox11 are maternally expressed at the animal pole (arrows). B,B’: During gastrulation, sox4 and sox11 are expressed in the expanding mesoderm surrounding the blastoporus (arrows). Dotter lines indicate the level of sections shown in C,C’. C,C’: Sections through embryos at stage 10.5 indicate a strong expression in the mesoderm (m). Ectoderm (ec) and endoderm (en) are negative for sox4 and sox11. bc = blastocoel. D,D’: At stage 15, sox4 and sox11 are strongly expressed in the anterior neural tissue (white arrowheads). In addition, sox4 transcripts are visualized in the cardiac progenitors (red arrowhead) whereas sox11 is detected at the placodal primordium (white arrow). E,E’: At stage 20, sox4 and sox11 are expressed in the neural tube (black arrows), the forebrain (green arrowheads), the eye anlagen (white arrowheads), the migrating neural crest cells (white arrows) and cardiac progenitor cells (red arrowheads). F,F’: Sox4 and sox11 mRNA molecules are detected in the neural tube (black arrows), the eye (white arrowheads) and the migrating neural crest cells (white arrows). The dotted line indicates the level of section shown in K and K’. G,G’: At stage 27, sox4 and sox11 are expressed in the hindbrain (black arrows), the eye (white arrowheads), the forebrain (green arrowheads), and the cardiac region (red arrowheads). H,H’: Sox4 and sox11 are strongly expressed in the hindbrain (black arrows) and branchial arches (white arrows). The dotted line indicates the level of section shown in L and L’. I,I’: Sox4 and sox11 transcripts are visualized in the hind- and forebrain (black and green arrows) as well as branchial arches (white arrows). Sox4 is additionally visualized in the vitelline veins (red arrow). Dotted line indicates the level of section shown in M and M’. J,J’: At stage 41, sox4 and sox11 are expressed in the head mesenchyme (white arrows), the forebrain (green arrowheads) and migratory primordium of the lateral line system (m). K–N,K’–N’: Transverse sections at indicated stages. K,K’: Sox4 and sox11 are detected in the optic vesicle (white arrowheads). Note that expression is missing from the optic stalk (os). L: Sox4 transcripts are detected in the retina (yellow arrow), the cornea epithelium (Ce) and the hindbrain (black arrow). In addition, sox4 is expressed in the placodes and the ganglia of the profundal (pPr; gPr) and trigeminus (pV; gV) nerve as well. Note that the placodal expression is only in the deep layers whereas the superficial layer is quite free of staining. L’: Sox11 expression is detected in the brain (black arrow), strongly in the ganglion cell layer of the retina (white arrowhead) with a gradient towards outer retinal layers. M: Sox4 transcripts are visualized in the retina (yellow arrow), the corneal epithelium (Ce), the mid- (black arrow) and forebrain (green arrowhead). A strong expression in the profundal (gPr) and trigeminus (gV) ganglia is shown. M’: Sox11 mRNA is visualized in the differentiated dorsal interneurons of the neural tube (black arrow) and the retina with a strong expression in the ganglion cell layer (white arrowhead). N, N’: Sox4 and sox11 are expressed in the ciliary marginal zone (CMZ) of the eye
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