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Early subdivisions in the neural plate define distinct competence for inductive signals

Daisuke Kobayashi1, Makoto Kobayashi2, Ken Matsumoto3, Toshihiko Ogura3, Masato Nakafuku1 and Kenji Shimamura1,*

1 Department of Neurobiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
3 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan



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  		Fig. 1. Induction of En2 by FGF signaling. Dissected brains from HH19-21 chick embryos whole-mount immunostained for En2. Electroporation (EP) was done at HH8 with GFP only (A), constitutively active Fgfr3 (B), constitutively active Fgfr1 (C) and Fgf8b (E) in the fore-midbrain region. Areas of the transgene expression were monitored by expression of GFP co-electroporated (D,F). Ectopic En2 expression is detected in the posterior diencephalon (arrows in B,C), and bordered by the zona limitans intrathalamica (ZLI; dashed lines in C), although the transgenes are expressed more anteriorly (arrowheads in D,F). Bars, 0.5 mm. di, diencephalon; is, isthmus; me, mesencephalon; os, optic stalk; rh, rhombencephalon; te, telencephalon.

 


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  		Fig. 2. Irx3 provides the posterior competence for Fgf8. Distribution patterns of Irx3 mRNA (A) and its protein (B) in HH21 embryo. (C) Two-color in situ hybridization for Irx3 (purple) and Shh (red) in an HH19 dissected brain. The anterior border of the Irx3-expressing domain is delineated by the ZLI in which Shh is expressed (arrows in A-C). HH16-21 dissected brains were immunostained for En2 (D,G,H-K) or hybridized for Fgf8 (F,L). Electroporation was done at HH8 with Irx3 (D-F), Irx3 and constitutively active Fgfr1 (G), dominant-negative Fgfr3 (H), Irx3 and dominant-negative Fgfr3 (I), Pax2 (J,L) and Pax2 with dominant-negative Fgfr3 (K). The expression vectors for the dominant-negative Fgfr3 and Irx3 or Pax2 were mixed at 9:1 prior to injection. H is an oblique posterior view of the specimen, and the dorsal midline is indicated by dashed lines in H and K, highlighting that the right side (electroporated) of the midbrain region has shrunk and the expression of En2 is severely downregulated. Note that ectopic En2 expression (arrowheads in D) is found only in the vicinity of the Fgf8-expressing sites (arrowheads in F), despite broad expression of the transgenes as visualized by GFP fluorescence (E). Bars, 0.5 mm.

 


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  		Fig. 3. Six3 confers the anterior competence for Fgf8. Dissected brains from HH19 embryos that had been electroporated at HH8 with GFP (control; A) and Fgf8b (B) were in situ hybridized for Bf1. (C) Normal expression of Six3 in a HH19 dissected brain. Ectopic Bf1-expressing domains are detected in the hypothalamic region (arrow in B). Dissected brains from HH13 embryos were stained for Bf1 (D, control; E, Six3-misexpressed) and for Fgf8 (F, normal embryo). Note an ectopic Bf1-expressing domain at the mid-hindbrain boundary where Fgf8 is expressed (arrows in E,F). Embryos were cultured by New’s method and electroporation was done at HH5. Note the efficient introduction of exogenous genes into the entire brain (E'). (G,H) HH25 dissected brain to which Six3 with constitutively active Fgfr1 (G,G') or Fgfr3 (H) were introduced at HH10, stained for Bf1 (G,G') and Emx2 (H). The specimen was flat-mounted to expose the anterior hindbrain region, showing the experimental side on the left (H). Ectopic Bf1- or Emx2-expressing cells are detected (arrows in G,G',H). Bars, 0.5 mm. is, isthmus; me, mesencephalon; ov, optic vesicle; pr, prosencephalon; rh, rhombencephalon; vm, ventral midline.

 


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  		Fig. 4. Six3 and Irx3 alter the regional responses to Shh. HH21 dissected brains normal (A,C,F), Irx3- (B,D,E), Six3- (G) and Six3 and Shh-misexpressed (H), were in situ hybridized for Nkx2.1 (E-H) or Nkx6.1 (A,B), or immunostained for Hnf3ß (C,D). Ectopic expression of Nkx2.1, Nkx6.1 and Hnf3ß, and the repression of Nkx2.1, are indicated by arrowheads (B,D,G,H) and an arrow (E), respectively. The sites of transgene expression were detected by GFP fluorescence (B',D',E',G',H'). Bars, 0.5 mm.

 


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  		Fig. 5. Misexpression of Irx3 resulted in an ectopic tectum formation in the forebrain. (A) Dorsal view of a dissected brain of a HH39 embryo that had been electroporated with Irx3 in the forebrain region at HH11. A small bulge is visible on the right side (electroporated) of the diencephalic region (arrow in A). (B) Thionine staining and (C-E) immunostaining for Pax7 of coronal sections of the ectopic bulge (B-D) and the normal tectum (E). (D,E) High-power views of the sections, in which the ventricle is located at the bottom of the panels. The ectopic bulges exhibit layered organization (arrow in B), and expression of Pax7 in both the ventricular zone (arrow in C,D) and some upper layers of cells (bracket in D), which resembles the optic tectum (arrow and bracket in E). Bars, 0.5 mm. ce, cerebellum; my, myelencephalon; ot, optic tectum; te, telencephalon.

 


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  		Fig. 6. Mutually repressive interactions establish segregated domains in the anterior neural plate. (A-C) Early expression of Six3 (A, red staining in C), Irx3 (B, blue staining in C) in HH6 chick embryos revealed by whole-mount in situ hybridization. The anterior side of the embryos is up. (D-F) Dissected brains from HH13 embryos in situ hybridized for Six3 (D), Irx3 (E), and Pax6 (F). Note that the anterior border of Irx3 expression (arrow in E) is located anterior to the posterior boundary of the Pax6-expressing domain (arrow in F). (G-J) Six3 (G,G'') or Otx2 (H) expression in presumptive HH10 embryos that had been electroporated with Irx3 at HH4. HH21 dissected brains that had been electroporated with Six3 (I,I') and ADSix3 (J,J') at HH8 stained for Irx3. Dorsal (G,G',H,H'), frontal (G''), and lateral views (I-J') of the specimens. Arrows indicate sites where Six3 or Irx3 expression was suppressed (G,G'',I',I''), and arrowheads mark ectopic expression of Irx3 in the ventral thalamus (J,J'). Asterisk in I indicates a crack in the specimen. The ZLI is represented by dashed lines in J. The expression of transgenes is monitored by GFP fluorescence (G',H',I''). Bars, 0.5 mm (A-H), 0.25 mm (I',I'',J'). hf, head fold; nc, notochord; ps, primitive streak.

 


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  		Fig. 7. A model for the early regionalization of the chick anterior neural plate. During HH4-6, a system that involves Otx2 and Gbx2 sets up domains and a boundary corresponding to the prospective mid-hindbrain boundary where the isthmic organizer (isO) develops (A) (Broccoli et al., 1999; Millet et al., 1999; Katahira et al., 2000). At around the same time, mutual repression between Irx3 and Six3 positions a boundary within the prospective forebrain, which later coincides with the ZLI (B). Slightly later, mutual repression between Pax6 and Pax2/En1 defines an intermediate boundary between the above two that corresponds to the dien-mesencephalic boundary (C) (Matsunaga et al., 2000). As these systems function independently, the early anterior neural plate accordingly acquires three boundaries at different anterior-posterior levels, being subdivided into four discrete domains potentially by a combinatorial code of transcription factors (D). Subsequently, localized signals produced from various signaling centers (e.g. anr, isO, pcp, nc) create fields of distinct histogenetic properties (e.g. te, ht, ot, ce; E). The longitudinal axes of the neural plate and tube are indicated by red arrows. anr, anterior neural ridge; ce, cerebellum; di-mes, dien-mesencephalic boundary; ht, hypothalamus; isO, isthmic organizer; nc, notochord; ot, optic tectum; pcp, prechordal plate; te, telencephalon; zli, zona limitans intrathalamica.

 

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