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First published online 19 December 2007
doi: 10.1242/dev.010082

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Six3 inactivation causes progressive caudalization and aberrant patterning of the mammalian diencephalon

Alfonso Lavado^*, Oleg V. Lagutin^* and Guillermo Oliver

Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794, USA.

View larger version (117K): [in this window] [in a new window]   Fig. 1. The anterior region of the Six3-null mouse brain progressively caudalizes during development. (A-G') At the 15- to 17-somite stage, (A) Irx1 is expressed in the mesencephalon, pretectum and thalamus of the wild-type brain, (B) Lhx5 in the prethalamus and the telencephalon, (C) Arx in the prethalamus and dorsal telencephalon, (D) Fezf1 and (E) Fezf2 in the ventral prethalamus and telencephalon, (F) Nkx2.1 in prethalamus and hypothalamus, and Six6 (G) in the Rathke's pouch (arrow) and anterior hypothalamus. (H-K') At the 15- to 22-somite stages, Irx3 is normally expressed in the mesencephalon, pretectum and thalamus (H,I). At the 19- to 22-somite stages, Tcf4 is normally expressed in the pretectum, thalamus (bracket) and ventral prethalamus (arrow) (J,K). At these somitic stages, the anterior-most part of the Six3-null brain has not fully posteriorized, as indicated by the anteriorly expanded Irx1 expression (A', arrowhead) and lack thereof in the dorsal brain (arrow), the presence of Lhx5 (B') and the absence of Irx3 and Tcf4 expression (H',J', brackets). Reduced Nkx2.1 expression (F') and the absence of Six6 (G') confirmed the absence or reduction of the hypothalamus in the Six3-null brain. Residual Six6 expression in surface ectoderm at the 15-somite stage suggested the presence of the Rathke's pouch region (arrow); however, no such structure is present at later stages. Later, the alar plate of the Six3-null brain gradually caudalizes, and at the 22- to 23-somite stage, the expression of Irx3 and Tcf4 (I',K', brackets) is anteriorly expanded. (L-N') At the 17-somite stage, an abnormally large number of TUNEL+ cells were seen in the anterior region of the Six3-/- brain (L'), as compared with wild type (L). Similar results were observed when the TUNEL assay was performed on sections of wild-type (M) and Six3-/- (M') brains. The plane of the tissue sections shown in M,M' is indicated by a line in L,L'. A drastic reduction in the number of apoptotic cells was observed in the anterior region of the Six3-null brain between the 17- and 21-somite stages (M',N'). (N) Bar chart showing the number of TUNEL+ cells per 100 DAPI+ cells at the 17-somite stage; white bar, wild-type; black bar, Six3-null. Arrows in B,C,D indicate the dorsal limit of expression. Anterior is to the right.

View larger version (135K): [in this window] [in a new window]   Fig. 2. The prospective thalamus is the most anterior structure of the Six3-null brain at the 22- to 24-somite stage. (A) Wtn3a is normally detected in the roof and alar plate (arrow) of the thalamus at the 22- to 24-somite stage; Fgf15 (B) and Otx1 (C) are localized in the alar plate of the thalamus (arrows) and prethalamus. In Six3-null mouse embryos, the prospective thalamus is located in the anterior region of the brain, as indicated by the expression of Wnt3a (arrow, A'), Fgf15 (arrow, B') and Otx1 (arrow, C'). The prospective prethalamus, as shown by Fezf1 (D) and Dlx2 (E) (arrows), is reduced in the Six3-null brain (arrows, D',E'). The alar plate-basal plate border, as indicated by Nkx2.2 expression (F), is also reduced (arrow, F'; the arrow in F shows the equivalent region to F'). Bmp4 is normally expressed in the basal plate of the posterior hypothalamus (arrow, G) and is absent in the Six3-null brain (G'). In the wild-type basal plate, Wnt1 is expressed in the ventral midline (H), and Ngn2 (I) and Foxa2 (J) are expressed more laterally in the tegmentum. The expression of these genes is expanded anteriorly in the Six3-null brain (H',I',J'). Arrows in H-J' indicate the most-anterior limit of the expression domain. The size of the mesencephalon, as indicated by the Fgf15 expression domain (B), and the region between the Pax6+ and En1+ territories (K, bracket), are normal in the Six3-null brain (B',K'). Schemes of the 22- to 24-somite stage wild-type (L) and Six3-null (L') brain. The pretectum, thalamus and prethalamus are located dorsally to the hypothalamus in the wild-type brain. In the Six3-null brain, the thalamus is the most anterior structure, and only a small portion of the prethalamus remains at this stage. The basal plate is shown in blue. Lines delimit the prospective regions. Anterior is to the right. HY, hypothalamus; PT, prethalamus; PTC, pretectum; T, thalamus.

View larger version (91K): [in this window] [in a new window]   Fig. 3. The prospective thalamus is replaced by the pretectum in the Six3-null brain. Hematoxylin and Eosin staining of sagittal sections from E14.5 wild-type (A) and Six3-null (A') mouse brains. At E14.5, Tcf4 is expressed in the pretectum and thalamus of wild-type embryos (B) and in the anterior-most region of the Six3-null brain (B'). At E13.5, Lhx2 is normally expressed in the thalamus (C), but is absent from the Six3-null brain (C'). Lhx2 is also expressed between the tegmentum and pretectum of the wild-type brain (arrow, C,C'). At E14.5, Lim1 is normally expressed in the pretectum (D), but is shifted anteriorly in the Six3-null brain (D'). Ebf1 is expressed in the anterior pretectum of wild-type embryos (E) and in the anterior region of the Six3-null brain (E'). As indicated by the absence of Dxl2 (F') and Nkx2.1 (G') expression at E14.5, the prethalamus (F) and the hypothalamus (G) were not present in the Six3-null brain. Instead, as indicated by Nkx6.1 expression at E12.5, the tegmentum is located in the ventral region of the mutant brain (H', arrows point to the limit of Nkx6.1 expression; compare with H). The ZLI, as revealed by Shh expression at E12.5, is not present in the Six3-null brain (I'; compare with I). Therefore, instead of the thalamus (J), the pretectum and the tegmentum are present in the anterior region of the Six3-null brain (J'). Anterior is to the right. GE, ganglionic eminence; HY, hypothalamus; P, pons; PT, prethalamus; PTC, pretectum; T, thalamus; TG, tegmentum.

View larger version (100K): [in this window] [in a new window]   Fig. 4. Six3 is not necessary for the expansion or maintenance of the ZLI. (A) At E12.5, Six3 is normally expressed in the tegmentum, prethalamus and hypothalamus. (B-D) Shh, Lim1 and Ngn2 (arrows in B,C and D, respectively) expression reveal the location of the ZLI. Lim1 is also expressed in the pretectum and Ngn2 in the thalamus. (E) Dlx2 is another marker for the prethalamus. Similar analyses were performed in Six3F/-;Six3-Cre (F-J), Six3F/-;CAGG-CreERT2 (TM-induced at E9.5; K-O) and Six3F/-;CAGG-CreERT2 brains (TM induced at E10.5; P-T). A similar efficiency of Six3 deletion was found in all three genotypes (F,K,P). As indicated by the expression of Shh (G,L,Q), Lim1 (H,M,R) and Ngn2 (I,N), the ZLI (arrows) was formed normally in these mutant mouse embryos. The thalamus, as indicated by Ngn2 expression (I,N,S), and the prethalamus, as shown by Dlx2 staining (J,O,T), were present in all of the conditional mutants. Asterisks indicate the telencephalon. Anterior is to the right. HY, hypothalamus; PT, prethalamus; PTC, pretectum; T, thalamus; TG, tegmentum.

View larger version (113K): [in this window] [in a new window]   Fig. 5. Prethalamus but not telencephalon is rescued in Six3-/-;Wnt1-/- embryos. Foxg1 is expressed in the telencephalon of wild-type (A) and Wnt1-/- (A') mouse brains at E10.5. No telencephalic expression of Foxg1 was observed in Six3-/- (A'') or Six3-/-;Wnt1-/- brains (A'''). Emx1 is expressed in the dorsal telencephalon of wild-type (arrow, B) and Wnt1-/- (arrow, B') brains. Tbr1 is expressed in the telencephalon (arrow) and eminentia thalami (arrowhead) of wild-type (C) and Wnt1-/- (C') brains. No telencephalic expression of Emx1 and Tbr1 was observed in the Six3-/- (B'',C'') and Six3-/-;Wnt1-/- (B''',C''') brains. Irx1 is expressed in the alar plate of the thalamus (arrow) of wild-type (D), Wnt1-/- (D') and Six3-null (D'') brains at E10.5. In the Six3-null brain (D''), Irx1 expression expands anteriorly; this ectopic expansion was rescued in the Six3-/-;Wnt1-/- brain (D'''). Similar results were obtained with Tcf4, a gene that is expressed in the alar plate of the pretectum and in the thalamus of wild-type (E), Wnt1-/- (E'), Six3-/- (E'') and Six3-/-;Wnt1-/- embryos (E'''). The prethalamus (arrow), as indicated by Fezf1 expression in the wild-type (F) and Wnt1-/- (F') brain, is extremely reduced in the Six3-null brain (F''); however, it appears normal in the Six3-/-;Wnt1-/- brain (F'''). A model for the Six3-/-;Wnt1-/- brain at this stage is shown (G'''). In the Wnt1-/- brain (G'), the mesencephalon is smaller than in the wild-type brain (G). The prospective thalamus is the most anterior structure in the Six3-null brain (G''), and the prethalamus remains in the Six3-/-;Wnt1-/- brain (G''') at this stage. The basal plate is indicated in blue. Lines delimit the prospective regions. Anterior is to the right. H, hindbrain; HY, hypothalamus; M, mesencephalon; PT, prethalamus; PTC, pretectum; T, thalamus; TEL, telencephalon.

View larger version (48K): [in this window] [in a new window]   Fig. 6. ZLI is rescued in the Six3;Wnt1 double-null brain. Hematoxylin and Eosin staining of sagittal sections from E14.5 Wnt1-null (Six+/+;Wnt1-/-, A) and Six3-/-;Wnt1-/- (A') mouse brains. As indicated by Shh expression, the ZLI (arrow) is normally detected in the alar plate of Wnt1-/- (B) and Six3-/-;Wnt1-/- brains (B'). Lhx2 and Tcf4 expression is detected in the thalamus of Wnt1-/- (C,D) and Six3-/-;Wnt1-/- (C',D') brains. BF-2 and Dlx2 labeled the prethalamus, indicating the presence of these structures in the Wnt1-/- (E,F) and Six3-/-;Wnt1-/- (E',F') brains. As revealed by Dlx2 and Nkx2.1 analyses, the hypothalamus was also present in Wnt1-/- (F,G) and Six3-/-;Wnt1-/- (F',G') brains at E14.5. Models representing the Six3+/+;Wnt1-/- (H) and Six3-/-;Wnt1-/- (H') brains at this stage are included for clarity. The thalamus and prethalamus are rescued in Six3-/-;Wnt1-/- brains (H'). Anterior is to the right. ET, eminentia thalami; HY, hypothalamus; P, Pons; PT, prethalamus; T, thalamus.