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First published online 13 September 2006
doi: 10.1242/dev.02585

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Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions

Tsutomu Hirata^1,*,, Masato Nakazawa^1,*, Osamu Muraoka¹, Rika Nakayama², Yoko Suda³ and Masahiko Hibi^1,

¹ Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.
² Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.
³ Vertebrate Body Plan Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.

View larger version (142K): [in a new window]   Fig. 1. Morphology of the forebrain of wild-type, Fez-/-Fezl+/-, Fez+/-Fezl-/-, Fez-/-Fezl-/- and Fez+/-Fezl+/- mice. Coronal (A-D,I-L) and sagittal (E-H) sections of E15.5 (A-H), E17.5 (I-L) wild-type (A,E,I), Fez-/-Fezl+/- (B,F,J), Fez+/-Fezl-/- (C,G,K), and Fez-/-Fezl-/- (D,H,L) mice. (M-Q) Sagittal sections of E18.5 Fez+/-Fezl+/- (M-O) and Fez-/-Fezl-/- (P,Q) mice at the level where posterior commissures (M,P, indicated by white arrowheads), habenulo-interpeduncular tracts (N,Q, white arrows) or mammillthalamic tracts (O, black arrowhead) were observed. Nissl staining. Fez-/-Fezl+/- mice had a very small olfactory bulb and Fez+/-Fezl-/- showed a reduction of the dentate gyrus (arrow in K,L), compared with wild-type littermates. Fez-/-Fezl-/- mice showed loss of the olfactory bulb (arrow in H), dentate gyrus and the CA3 region of hippocampus (arrow in L), prethalamus, strong reduction of the thalamus (region between arrowheads) and a reduced neocortex. At E18.5, both Fez+/-Fezl+/- and Fez-/-Fezl-/- mice had a posterior commissure and habenulo-interpeduncular tract. The habenulo-interpeduncular tracts were small and abnormally located, due to severe reduction of the thalamus in Fez-/-Fezl-/- mice. Fez+/-Fezl+/- (O), but not Fez-/-Fezl-/- mice, had mammilothalamic tracts. DG, dentate gyrus; hit, habenulo-interpeduncular tract; hip, hippocampus; ht, hypothalamus; ma, mamillary region; mtt, mammilothalamic trancts; ncx, neocortex; OB, olfactory bulb; pc, posterior commissure; th, thalamus; pt, pretectum; pth, prethalamus; s, septum; 3v, third ventricle. Scale bars: 0.5 mm.

View larger version (137K): [in a new window]   Fig. 2. Defects in prethalamus and thalamus development and rostral expansion of the pretectum in Fez-/-Fezl-/- embryos at E12.5. A-T) Expression of Dlx1 (marker for prethalamus and ganglionic eminence, A-D), Gbx2 (thalamus, E-H), Lhx1 (posterior pretectum and ZLI, I-L), Ebf1 (anterior pretectum, M-P) and Sox14 (rostral domain of thalamus, Q-T) in control, Fez-/-Fezl+/-, Fez+/-Fezl-/- and Fez-/-Fezl-/- embryos was analyzed by in situ hybridization. Sagittal sections with anterior to the left. Dlx1 expression was not detected in the prethalamus but was maintained in the hypothalamus and ganglionic eminence in Fez-/-Fezl-/- embryos (D). Gbx2 expression was strongly reduced and detected in patches (arrows in H) in Fez-/-Fezl-/- embryos. Lhx1 expression was absent in the ZLI, but was not significantly affected in the posterior pretectum of Fez-/-Fezl-/- embryos (I-L). The expression domain of Ebf1 was expanded rostrally in Fez-/-Fezl-/- embryos (P), compared with that in the wild-type, Fez-/-Fezl+/- and Fez+/-Fezl-/- littermates (M-O). The expression of Sox14 was abolished in Fez-/-Fezl-/- embryos (T). apt, anterior pretectum; GE, ganglionic eminence; ht, hypothalamus; ppt, posterior pretectum; pth, prethalamus; th, thalamus.

View larger version (95K): [in a new window]   Fig. 3. Defects in telencephalon formation and regionalization of the diencephalon in Fez-/-Fezl-/- embryos. Expression of Emx2 (A-D), Pax6 (E-H), Dlx1 (I-L), Shh (M-P), Tcf4 (Q-T) and Lhx5 (U-X) in control (A,E,I,M,Q,U), Fez-/-Fezl+/- (B,F,J,N,R,V), Fez+/-Fezl-/- (C,G,K,O,S,W) and Fez-/-Fezl-/- embryos (D,H,L,P,T,X) at E10.5. (A-T) Whole-mount in situ hybridization and lateral views of anterior neuroectoderm, with anterior to the left. (U-X) Coronary sections of the diencephalon. Emx2 expression in the dorsal telencephalon was reduced in Fez-/-Fezl-/- embryos. Pax6 expression was downregulated in the thalamus at this stage (marked by dots, E,F,G) in control, Fez-/-Fezl+/- and Fez+/-Fezl-/- embryos, but not in Fez-/-Fezl-/- embryos (arrow, H). The expression domain of Pax6 in the dorsal telencephalon was reduced but not significantly affected in the diencephalon except for the thalamus (region between arrowheads) in Fez-/-Fezl-/- embryos. Dlx1 expression was detected in the ganglionic eminence (arrow, I,L) but not in the prethalamus (arrowhead, I,L) in Fez-/-Fezl-/- embryos. Shh expression was detected in the AEP, but not in the ZLI in Fez-/-Fezl-/- embryos (arrow, P), compared with controls (M-O). The domain expressing Tcf4 at a high level (thalamus and pretectum region, marked by dots) was expanded in Fez-/-Fezl-/- embryos (T), compared with controls (Q-S). Lhx5 expression was abolished in the prethalamus of Fez-/-Fezl-/- embryos (X). ht, hypothalamus; pth, prethalamus; th, thalamus.

View larger version (63K): [in a new window]   Fig. 4. Reduction of rostral diencephalon and expansion of caudal diencephalon in Fez-/-Fezl-/- embryos at E9.5. Expression of Emx2 (A,B), Pax6 (C,D), Fgf8 (E,F), Wnt3a (G,H) and En2 (I,J) in control (A,C,E,G,I) and Fez-/-Fezl-/- embryos (B,D,F,H,J) was analyzed by whole-mount in situ hybridization. Lateral views of anterior neuroectoderm, with anterior to the left. Expression of Emx2 (A) and Pax6 in the dorsal telencephalon (between arrowheads, C) was reduced, but the Pax6 expression in the diencephalon (marked by dots) was maintained in Fez-/-Fezl-/- embryos. Fgf8 expression in the commissural plate and mid-hindbrain boundary was maintained, but that in the dorsal prethalamus was absent (arrowheads E,F) in Fez-/-Fezl-/- embryos. The expression domain of Wnt3a was caudal to the ZLI in control embryos (G) and was expanded rostrally in Fez-/-Fezl-/- embryos (arrow in H). Expression of En2, which marks the midbrain, was not affected in Fez-/-Fezl-/- embryos (I,J). cp, commissural plate; mhb, mid-hindbrain boundary; pth, prethalamus.

View larger version (68K): [in a new window]   Fig. 5. Rostrally expanded expression of Irx1 in Fez-/-Fezl-/- embryos. Irx1 and Six3 expression in control (A,C,E,G,I) and Fez-/-Fezl-/- (B,D,F,H,J) embryos at E10.0 (A,B), E9.5 (C,D,I,J), E9.0 (E,F) and E8.5 (G,H). Lateral views, with anterior to the right. Irx1 expression was not significantly different between Fez-/-Fezl-/- and control embryos at E8.5. A rostral expansion of Irx1 expression was detected at E9.0 in Fez-/-Fezl-/- embryos (arrow). At E9.5 and E10.5, the rostral limit of Irx1 expression (arrows) reached the caudal edge of the telencephalon (marked by dots). Expression of Six3 was not significantly affected in Fez-/-Fezl-/- embryos at E8.5 (data not shown) and at E9.5 (I,J).

View larger version (115K): [in a new window]   Fig. 6. Complementary expression of Fez/Fezl and Irx1. (A-D) Expression of Fez (A,D), Fezl (B) and Irx1 (C,D) at E8.5. (D) Two-color staining. Fez and Irx1 transcripts were stained with BM Purple and Fast Red; the fluorescence image from the Fast Red was superimposed on the bright-field image. (E-M) Expression of Fez (E), Fezl (F) and Irx1 (G) at E9.5. (H-M) Sagittal sections of E9.5 embryos were hybridized with Fez and Irx1 (H-J), or Fezl and Irx1 probes (K-M). The hybridized signals were stained with BM Purple (Fez and Fezl) and Fluorescein (Irx1). Bright-field images (H,K), fluorescence images (I,L) and the bright-field and fluorescence superimposed images (J,M).

View larger version (28K): [in a new window]   Fig. 7. Enhancer and promoter region of Fezl. (A) Schematic diagram of the Fezl enhancer/promoter and the constructs used for transgenesis. (B-D) Detection of lacZ expression by X-gal staining in Fezl8.2p-lacZ (B) and Fezl2.7p-lacZ (C,D) transgenic mouse embryos, in which lacZ expression was driven by the 8.2 kbp and 2.7 kbp Fezl enhancer/promoter, respectively. (B,C) E8.5 embryos, lateral views with anterior to the top. (D) E9.5 embryo, lateral views of the anterior neuroectoderm. The 8.2 kbp Fezl enhancer/promoter recapitulated the endogenous Fezl expression (rostral to the ZLI), whereas the 2.7 kbp Fezl enhancer/promoter showed a caudally expanded expression of lacZ (arrows, D).

View larger version (46K): [in a new window]   Fig. 8. Misexpression of Fez or Fezl affects rostro-caudal polarity in the diencephalon. Misexpression of Fezl (A-Q) or Fez (R-U) by the 2.7 kbp Fezl enhancer/promoter (Fezl2.7p-Fezl-IRES-Venus, Fezl2.7p-Fez-IRES-Venus), or Fezl by the FM enhancer of the Otx2 gene and mouse Hsp68 promoter (Otx2FM-Hsp68-Fezl-IRES-Venus, V,W) affected the diencephalon subdivisions. Exogenous Fezl expression was monitored by the expression of Venus attached to an IRES (Fig. 7) (fluorescence images, A,D,G,J,L,O,R,T,V). E9.5 embryos were analyzed by whole-mount in situ hybridization with Foxg1 and Irx1 (B,C,S,W). E10.5 embryos were analyzed by in situ hybridization with Tcf4 (E,F,U) or Shh (H,I,K). Sagittal sections of E12.5 embryos were analyzed with Dlx1 or Gbx2 probes (M,N,P,Q). (C,F,I,N,Q) Control non-transgenic embryos. Expression of Foxg1 in the telencephalon was not affected (caudal limit marked by arrows), but the rostral limit of Irx1 expression (marked by arrowheads) was shifted caudally in Fezl2.7p-Fezl-IRES-Venus (B), Fezl2.7p-Fez-IRES-Venus (S) and Otx2FM-Hsp68-Fezl-IRES-Venus embryos (W), compared with the control (C). Tcf4-high expression domain in the thalamus and prethalamus was strongly reduced in the Fezl2.7p-Fezl-IRES-Venus (E) and Fezl2.7p-Fez-IRES-Venus (U) embryos. Shh expression in the ZLI (arrows) and ventral diencephalon was reduced (H, n=2/4), or Shh expression in the ZLI was shifted caudally (K, n=2/4) in the Fezl2.7p-Fezl-IRES-Venus embryos, compared with control (I). Dlx1 expression in the prethalamus was expanded caudally when the exogenous Fezl-IRES-Venus was strongly expressed (M, n=1/2). Gbx2 expression in the thalamus was strongly reduced in the Fezl2.7p-Fezl-IRES-Venus embryos (P, n=2/3).

View larger version (15K): [in a new window]   Fig. 9. Schematic presentation of a role of Fez and Fezl in diencephalon patterning. Fez and Fezl are expressed in the telencephalon and rostral diencephalon (prospective prethalamus) and function to suppress the formation of the caudal diencephalon, which expresses Irx1 and Wnt3a. In the absence of Fez and Fezl, the rostral diencephalon does not form and instead caudal diencephalon expands rostrally at E9.5. Subsequently at E10.5, the prethalmaus and the ZLI, which is normally located in the interface between the prethalamus and thalamus, do not form in Fez-/-Fezl-/- embryos. The caudal diencephalon, including the thalamus and pretectum, is expanded in Fez-/-Fezl-/- embryos. The formation of thalamus, however, is dependent on inductive signals (e.g. Shh) from the ZLI. In Fez-/-Fezl-/- embryos, the thalamus does not grow properly, but the anterior pretectum remains expanded at E12.5. apt, anterior pretectum; di, diencephalon; pth, prethalamus, ppt, posterior pretectum; tel, telencephalon; th, thalamus.