September 1, 2009;
Xhairy2 functions in Xenopus lens development by regulating p27(xic1) expression.
of vertebrate eyes
is derived from competent pre-placodal ectoderm
in response to signal(s) from retinal lineage. We herein report that the Xenopus Hes gene Xhairy2
, which is expressed in pre-placodal ectoderm
, is required for lens
development from the initial stage. We show that Xhairy2
knockdown reduced the expression of lens
marker genes at every step of lens
determination, eventually resulting in ocular lens
malformation. Interestingly, retina
marker gene expression and retinal anlage morphology remained normal upon Xhairy2
knockdown. Furthermore, loss of lens
field caused by Xhairy2
depletion was partially rescued by simultaneous knockdown of the cell cycle inhibitor gene p27
(xic1). These results suggest that Xhairy2
is required for lens
development through the regulation of p27
(xic1) expression, independent of the known cascade of transcription factors. Based on these findings, we propose that Xhairy2
may maintain an intracellular environment in which inducing signal(s) can be accepted.
[+] show captions
Figure 1. Xhairy2 is expressed in PPE from gastrula stage. WISH analyses of Xhairy2 expression from mid to late gastrula stage in comparison with PPE-marked dlx5. a-i': Xhairy2 expression. a, d, g: Dorsal views with anterior side down. b, e, h: Anterior views with dorsal side up. White rectangles in b, e, and h indicate PPE expression. c, f, i: Mid-sagittal views with anterior side to the right. Embryos were halved and the left halves are shown. c', f', i': High-magnification views of areas enclosed by black rectangles in c, f, and i, respectively. White brackets indicate PPE expression in the sensorial layer, not in the superficial layer. j-r': dlx5 expression. Samples are shown in the same way as a-i'.
Figure 3. Effects of Xhairy2 knockdown on the expression of lens and retinal marker genes in tail-bud-stage embryos. Co MO (3.4 ng: a-f') or Xhairy2 MO (6.9 ng: g-l'), together with EYFP and lacZ mRNA, was micro-injected into one dorsal animal blastomere at the 8-cell stage, and the embryos were harvested at st. 25 (for six6), st. 28 (for notch2, foxE3, L-maf, gamma 1-cry), or st. 32 (for rx1), followed by WISH. MO-receiving cells were visualized by X-gal staining (green). Red arrowheads indicate reduction of marker gene expression. Note that Xhairy2 knockdown reduced the expression of LP marker genes, while virtually no effect was seen on the expression of retinal marker genes six6 and rx1.
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.
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.
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).
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').
Fig. 2. Xhairy2 knockdown caused malforma- tion of lens without affecting retina in terms of morphology. Co MO (3.4 ng) or Xhairy2 MO (6.9 ng), together with EYFP mRNA, was micro-in- jected into one dorsal animal blastomere at the 8-cell stage. The uninjected side served as con- trol. Injected embryos were harvested at st. 43 for histological analyses. a Co MO injected tadpoles. a: A representative tadpole. EYFP fluorescence indicates MO-receiving region. b: Immunohistochemistry of 1-crystallin pro- tein. Lenses were specifically stained brown. inj, the injected side. c, d: High-magnification views of lenses shown in b. c d High-magni- fication views of retinas shown in b. e Xhairy2 MO-injected tadpoles. Samples are shown in the same way as ad Red arrowhead in f indicates malformed lens that is still immu- nopositive for 1-crystallin. Note that the lay- ered pattern in the retina is maintained (h, although there seem to be a large number of cells in the nuclear layer and the ganglion layer. L, lens; g, ganglion layer; n, nuclear layer; p, photoreceptor cell layer.