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Gene/Clone	Species	Stage	Anatomy Item	Experimenter
pax6	xenopus

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Experiment details for pax6

Kiem LM et al. (2017) Assay

The Nedd4 binding protein 3 is required for anterior neural development in Xenopus laevis.

Gene	Clone	Species	Stages	Anatomy
pax6.L		laevis	NF stage 13	neuroectoderm , pre-chordal neural plate , chordal neural plate , neural plate , optic field , [+] anterior placodal area
pax6.L		laevis	NF stage 23	brain , forebrain , diencephalon , hindbrain , spinal cord , [+] optic vesicle , anterior placodal area

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Fig. 5. Loss of N4bp3 function interferes with the expression of eye and brain marker genes. A WMISH using rax and pax6 at stage 13 demonstrates a reduced staining in the eye field on the injected side (red arrows) whereas the un-injected and Control MO injected sides were unaffected. In contrast, the pan-neural maker gene sox3 is not affected upon N4bp3 (25 ng MO) deficiency. B Quantification of the data in A. C. Expression of pax6 in the brain region is reduced in N4bp3 deficient embryos at stage 13. D Statistical evaluation of the data in A. E The expression domain and intensity of rax, pax6 and otx2 in the eye region at stage 23 is impaired upon loss of N4bp3 function (black arrows). F Statistical evaluation of the data in E. G WMISH using emx1, pax6 (both forebrain expression domains), otx2 (forebrain and midbrain expression domains), en2 (isthmus expression domain) and egr2 (krox20) (hindbrain expression domain). All analyzed marker genes are significantly reduced upon loss of N4bp3 function (black arrows). Control MO injection has no effect on marker gene expression. H Quantification of the data in G. Abbreviations: N, number of evaluated embryos in total; n, number of independent experiments. Error bars indicate standard error of the means (s. e. m.); * p≤0.05; ** p≤0.001. P-values were calculated by a nonparametric, one-tailed Mann-Whitney rank sum test.

Gene	Clone	Species	Stages	Anatomy
pax6.L		laevis	NF stage 42	retina , eye , retinal ganglion cell layer , amacrine cell , retinal ganglion cell

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Fig. 2. N4bp3 is required for eye development in Xenopus laevis. A Knockdown of N4bp3 causes a severe eye phenotype demonstrated on macroscopic and microscopic level. The eye on the injected side is smaller and deformed (white arrows) including a coloboma phenotype (red arrows). Scale bars: dorsal and lateral: 500 µm; detail: 100 µm. B N4bp3 MO injection results in an eye phenotype in a dose dependent and significant manner. Note that the data of D are integrated into this statistical analysis. C The eye phenotype caused by 25 ng N4bp3 MO injection can significantly be rescued by co-injecting 1 ng full-length rat n4bp3 RNA. Scale bars: 1000 µm. D Statistical evaluation of the data in C. E Measurement of the area of the eye (red dotted circles) at stage 42. Scale bars: 500 µm. F The statistical evaluation of the data in E reveals a significant reduction of the eye area upon N4bp3 depletion. G and H Control MO embryos exhibit no colobma. Scale bar: 1000 µm. I and J Compared to internal control, N4bp3-deficient eyes show coloboma with variable severity. Scale bar: 1000 µm. K and L Loss of N4bp3 function leads to disorganized retinal layers including rosette-like structures (white arrows) shown by retinal cell type specific marker genes. However, retinal layers on the un-injected side are unaffected. We used rho (photoreceptor cells), prox1 (horizontal cells), vsx1 (bipolar cells), pax6 (amacrine and ganglion cells) and pouf4f1 (ganglion cells). Abbreviations: N, number of evaluated embryos in total; n, number of independent experiments; ng, nanogram. Error bars indicate standard error of the means (s. e. m.); * p≤0.05; ** p≤0.01; *** p≤0.001; **** p≤0.0001. P-values were calculated by a nonparametric, one-tailed Mann-Whitney rank sum test.