XB-ART-34621Development. May 1, 2005; 132 (10): 2401-13.
Six3 functions in anterior neural plate specification by promoting cell proliferation and inhibiting Bmp4 expression.
Although it is well established that Six3 is a crucial regulator of vertebrate eye and forebrain development, it is unknown whether this homeodomain protein has a role in the initial specification of the anterior neural plate. In this study, we show that exogenous Six3 can expand the anterior neural plate in both Xenopus and zebrafish, and that this occurs in part through Six3-dependent transcriptional regulation of the cell cycle regulators cyclinD1 and p27Xic1, as well as the anti-neurogenic genes Zic2 and Xhairy2. However, Six3 can still expand the neural plate in the presence of cell cycle inhibitors and we show that this is likely to be due to its ability to repress the expression of Bmp4 in ectoderm adjacent to the anterior neural plate. Furthermore, exogenous Six3 is able to restore the size of the anterior neural plate in chordino mutant zebrafish, indicating that it has the ability to promote anterior neural development by antagonising the activity of the BMP pathway. On its own, Six3 is unable to induce neural tissue in animal caps, but it can do so in combination with Otx2. These results suggest a very early role for Six3 in specification of the anterior neural plate, through the regulation of cell proliferation and the inhibition of BMP signalling.
PubMed ID: 15843413
PMC ID: PMC2789257
Article link: Development.
Grant support: 074376 Wellcome Trust , GGP04268 Telethon, TI_GGP04268 Telethon, WT074376 Wellcome Trust
Genes referenced: ascl2 bmp4 cdknx chrd.1 dhh elavl1 elavl3 foxg1 hes4 neurog1 neurog2 nppa otx2 pnp rax shh six3 sox2 tubb2b xk81a1 zic2
Morpholinos referenced: six3 MO1 six3 MO2 six3 MO3
Article Images: [+] show captions
|Fig. 1. Xsix3 promotes cell proliferation in the neural plate. (A-D) Transverse sections of stage 13 embryos injected with either Xsix3 (A,C) or VP16-Xsix3 (B,D), processed simultaneously for BrdU incorporation (brown nuclear staining), and Zic2 (A,C, blue staining) or lacZ (B,D, red staining) expression. Sections are at the level of the anterior (A,B) or posterior neural plate (C,D). ANP, anterior neural plate; PNP, posterior neural plate. (E,F) The average number of BrdU-positive cells per section in either the control side (magenta) or the side injected with Xsix3 (blue) or VP16-Xsix3 (green). Error bars indicate s.e.m. (G) Double whole-mount in situ hybridization of a stage 13 embryo shows that the Xsix3 (magenta) and Xbf1 (purple) expression domains overlap. (H-J) Stage 13 (H,I) and stage 14 (J) embryos injected with Xsix3 display expansion of the Xbf1 (H), Xrx1 (I) and cyclinD1 (J) expression domains. White brackets indicate the anterior expression domain of cyclinD1 in the control (right) and injected (left) side. (K,L) Xsix3 overexpression represses p27Xic1 both in the anterior, trigeminal ganglion (K, arrow) and in its posterior expression domain (L, arrow) in stage 13 embryos. (G-K) Frontal views, dorsal towards the top; (L) dorsal view, anterior towards the bottom. Red staining represents expression of a co-injected lacZ lineage tracer. The injected side of the embryos (to the left of vertical bars or dotted lines representing the midline) is indicated (inj).|
|Fig. 2. Alterations of the size of the neural plate in Xsix3 gain- and loss-of-function experiments. (A-H,K-Z) Embryos injected unilaterally with Xsix3 (A-H,K-N), GR-Xsix3 (O-R), VP16-Xsix3 (S-V) or MoXsix3 (W-Z), and analysed at early neurula stage. The probes used are indicated in each panel. Red staining in A,B,D-H,K-Z and turquoise staining in C represent expression of the co-injected lacZ lineage tracer. The injected side of the embryos (to the right of dotted lines representing the midline) is indicated (inj). (A-G,K-M,O-Z) Frontal views, dorsal towards the top; (H,N) ventral views (VV), anterior towards the bottom. (H,N) Arrows indicate Xk81 repression caused by Xsix3 overexpression. (I,J) Embryos untreated (I) or treated (J) with HUA, brown nuclear staining representing anti-phosphorylated histone H3 staining; dorsal views, anterior towards the bottom. (K-N) Embryos injected with Xsix3 and treated with HUA. (A,B,K) White brackets indicate the anterior expression of Sox2 (A,K) or Xnrp1 (B) in Xsix3-injected (right) and control (left) sides.|
|Fig. 5. Xsix3 and Bmp4 repress each other. (A-Q) Control and injected embryos, and animal caps, were analysed at stage 13 for the expression of the genes indicated. (A-C) Xsix3 overexpression represses Bmp4 expression in embryos (A) and animal caps (C), whereas uninjected caps (B) strongly express Bmp4. (D) Xsix3 is repressed in embryos injected with Bmp4. (E) tBR injection expands the Xsix3 expression domain. (G-I) Animal caps injected with tBR (H) or Chordin (I) strongly express Xsix3, whereas uninjected caps (G) do not express Xsix3. (J,K) MoXsix3- and VP16-Xsix3-injected embryos display anterior expansion of Bmp4 (the white dots indicate the anterior neural plate border). (L,M) VP16-Xsix3 and Bmp4 injection induces apoptosis (TUNEL staining) in the anterior neural plate. (N,O) MoXsix3 reduces (N) and tBR expands (O) the domain of Zic2 expression. (P,Q) Zic2 repression is rescued in embryos co-injected either with MoXsix3 and tBR (P, compare with N), or with MoXix3 and Xsix3 (Q, compare with N).|
|Fig. 3. Xsix3 regulates the expression of genes that control cell differentiation, and its expression is controlled by Hedgehog and Neurogenin signalling. (A-C) Embryos injected unilaterally with Xchh (A) and Xngnr1 (B,C) display ectopic expression of Xsix3 (A) and N-tubulin (B), and repression of Xsix3 (C). (D) Comparison of the expression of Xsix3 (light blue) with that of Xngnr1 (purple) in a stage 13 embryo. (E-K) Embryos injected with Xsix3 display repression of N-tubulin (E, the arrow indicates the repressed expression in the trigeminal ganglion), elrC (F) and Xngnr1 (J), and ectopic expression of Zic2 (G), Hairy2 (H) and Xash3 (I). (K,L) Embryos injected with Xsix3 and treated with HUA show expansion of Zic2 (K) and repression of N-tubulin (L). (M,O,P) Embryos injected with VP16-Xsix3 display reduction of the Xash3 (M), Zic2 (O) and Xhairy2 (P) expression domains. (J,N) Arrows indicate the anterior boundaries of Xngnr1 in the injected side of the embryo. (A-P) Frontal views, dorsal towards the top. Red staining in B,C,F-P and turquoise staining in A,E represent expression of the co-injected lacZ lineage tracer. The injected side of the embryos (to the left of dotted lines representing the midline) is indicated (inj). (H) White brackets indicate the anterior expression of Xhairy2 in Xsix3-injected (left) and control (right) sides.|
|Fig. 4. Xsix3 represses epidermal fate but requires Xotx2 for the induction of neural markers in animal caps. Animal caps from control uninjected embryos (Control) or embryos injected with either Xsix3, Xotx2, or a mixture of the two RNAs, as indicated at the top, were dissected at stage 9, cultured to stage 14 and analysed for the expression of the genes indicated to the left. The column to the right (Embryo) shows expression of the analysed genes in control embryos.|
|Fig. 6. six3 rescues chordin loss of function. (A-D) Lateral view of 24 hpf zebrafish embryos. Arrowheads indicate the eye/brain size in control (A), and in embryos injected with MoChd (B), six3 (C), or with both six3 and MoChd (D). Arrows indicate the posterior region, which is expanded in embryos injected with MoChd, or co-injected with MoChd and six3. (E-X) Early neurula embryos (90% epiboly) treated as in A-D and analysed for expression of the genes indicated. (E-P,U-X) Dorsal-anterior views, vegetal towards the bottom; (Q-T) lateral views, anterior towards the top.|
|Fig. 7. Xsix3 directly represses Bmp4 transcription. (A,B) Animal caps were analysed at stage 13 for the expression of the genes indicated to the left. (A) Bmp4 repression in GR-Xsix3-injected animal caps treated either with dexamethasone (DEX) alone, or with both DEX and cycloheximide (CHX), compare with control with no DEX treatment shown above. (B) Control of CHX treatment. Xotx2-GR-injected animal caps treated with DEX alone, or with both DEX and CHX. (C) Xsix3 binds to a Bmp4 promoter fragment containing homeodomain-binding sites. Xsix3-GST, but not GST alone, binds to the labelled Bmp4 promoter to form various complexes in a concentration-dependent manner. Addition of an excess of unlabelled Bmp4 promoter fragment (50- to 350-fold) significantly inhibits the binding. The arrow indicates the major complex formed with the highest protein concentration. The arrowhead indicates the free probe. The amount of added protein is indicated in micrograms.|