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Gene/Clone	Species	Stage	Anatomy Item	Experimenter
pax6	xenopus

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Experiment details for pax6

Murato Y and Hashimoto C (2009) Assay

Xhairy2 functions in Xenopus lens development by regulating p27(xic1) expression.

Gene	Clone	Species	Stages	Anatomy
pax6		xenopus	NF stage 12.5	pre-chordal neural plate , neural plate
pax6		xenopus	NF stage 15	pre-chordal neural plate , chordal neural plate , trigeminal placode , neural plate
pax6		xenopus	NF stage 15	trigeminal placode , neural plate , anterior neural fold , neural tube

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Figure 5. pax6 LF phenotype of Xhairy2 knockdown is partly caused by ectopic p27xic1 expression. Micro-injection, lineage tracing, and photography were performed as described in Figure 3. a, b: Analyses of X-MyT1 expression. X-gal staining was omitted to facilitate analyses. c, d: Analyses of p27xic1 expression. e, f: Two-color WISH analyses of pax6 (light blue) and p27xic1 (purple). g, h: WISH analyses of st.-15 embryos for visualization of LF (pax6 and six3) and neural crest (foxD3). Arrows indicate LF expression. i-l: Analyses of pax6 expression. Doses of MOs and mRNA are described in the text. Blue arrowheads indicate ectopic expression and red arrowheads indicate reduction of marker gene expression.

Gene	Clone	Species	Stages	Anatomy
pax6		xenopus	NF stage 15	pre-chordal neural plate , chordal neural plate , neural plate , anterior , neural tube

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Figure 4. Xhairy2 knockdown reduces the expression of LF marker genes in neurulae without marked changes in PPE marker expression. Micro-injection and lineage tracing were performed as described in Figure 3. Injected MO is indicated at the upper right corner and WISH probe is indicated at the lower right corner of each panel. a: Schematic presentation of LF in neurulae. b-i: LF marker genes. b, c: Two-color WISH of st.-15 embryos to confirm LF position. LF expression (black arrowheads) of pax6 (magenta) and six3 (purple) is indeed outside of the neural plate border that is marked by Xhairy2 (light blue). j, k: Embryos were injected with 6.9 ng of Xhairy2 MO and/or 20 pg of Xhairy2b mRNA where Xhairy2 MO does not bind in order to check the specificity of Xhairy2 MO to LF-loss phenotypes. I: Quantitative summary of j and k. Error bar: standard deviation of 5 independent experiments. m-t: PPE marker genes. Red arrowheads indicate reduction of marker gene expression. All the samples were pictured from the anterior side with the dorsal side up.

Gene	Clone	Species	Stages	Anatomy
pax6		xenopus	NF stage 15	lens placode , neural plate , anterior , neural tube

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Figure 7. Inhibition of cell cycle from gastrula to mid-neurula stage by HUA treatment does not affect the formation of LF and lens. a, b: BrdU detection to confirm effects of HUA. a', b': Transverse sections indicated by red lines in a, b. c: Counting of BrdU-positive cells. A representative result is shown. Error bar: standard deviation (n = 5). d-i': HUA treatment from st. 10+ to st. 15. d, e: LF expression of pax6 together with mitotic antigen PHH3. f, g: LF expression of six3. h-i': HUA-treated embryos after a wash from st. 15 showed less tail elongation (i) at st. 38 than control embryos (h). h', i': Transverse section of the embryos shown in h and i, respectively. Note that lenses were formed in HUA-treated samples (i'), although their sizes were smaller than that of control sample (h').

Gene	Clone	Species	Stages	Anatomy

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Figure 6. Xhairy2 knockdown affects proliferation within LF at late neurula stage, but not at mid-neurula stage. Micro-injection was performed as described in Figure 3. MO- or mRNA-injected embryos were further injected with BrdU and incubated for 1 hr prior to fixation at st. 16 (a-d) or st. 19 (e-h). To visualize LF, samples were stained with pax6 (a-d) or foxE3 (e-h) before BrdU detection. a-h: Whole-mount view from the anterior side with the dorsal side up. Red arrowheads indicate reduction of marker gene expression. a'-h': Horizontal sections of the corresponding samples for counting BrdU-positive cells within LF that is defined by LF expression of pax6 (a'-d') or foxE3 (e'-h'). Anterior side up. Red rectangles indicate LF or the corresponding region without marker gene expression. i: Quantitative summary of BrdU count. Five successive sections from each sample were subjected to counting of BrdU-positive cells within LF. The number of BrdU-positive cells from each section was summed per sample. Then, the proportion of BrdU-positive cells of the injected side with respect to the un-injected side was calculated per sample. The mean value of 3 independent samples is presented as the representative value. Error bar: standard deviation (n = 3).